Methods and compositions for generating or maintaining pluripotent cells

ABSTRACT

Methods and compositions are provided for generating or maintaining human iPS cells in culture. Methods include the use of a low osmolality medium to make human iPS cells, or use of a low osmolality medium to maintain human iPS cells. Methods for making targeted genetic modification to human iPS cells cultured in low osmolality medium are also included. Compositions include human iPS cells cultured and maintained using the low osmolality medium defined herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application No.62/064,384, filed Oct. 15, 2014, which is herein incorporated byreference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named469586SEQLIST.TXT, created on Oct. 14, 2015, and having a size of 758bytes, and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

BACKGROUND

Human induced pluripotent stem (iPS) cells can display a naïve or primedstate of pluripotency (Nichols and Smith, Cell Stem Cell (2009) Vol.4(6), pp. 487-492). Primed human iPS cells express characteristicssimilar to those of post-implantation epiblast cells, and are committedfor lineage specification and differentiation. By contrast, naïve humaniPS cells express characteristics similar to those of embryonic stem(ES) cells of the inner cell mass of a pre-implantation embryo. In somerespects, naïve iPS cells are more pluripotent than primed cells, asthey are not committed for lineage specification. Various cultureconditions can be used to maintain human iPS in a naïve state or in aprimed state.

SUMMARY

Methods are provided for making a population of human inducedpluripotent stem cells (hiPSCs). Such methods comprise culturing invitro a population of non-pluripotent cells, transformed to express apluripotent state, in a low osmolality medium comprising a base mediumand supplements, wherein the low osmolality medium comprises: (a) aleukemia inhibitory factor (LIF) polypeptide; (b) a glycogen synthasekinase 3 (GSK3) inhibitor; and (c) a MEK inhibitor; wherein the mediumhas an osmolality of about 175 mOsm/kg to about 280 mOsm/kg. Suchmethods can also comprise culturing in vitro a population ofnon-pluripotent cells, transformed to express a pluripotent state, in alow osmolality medium comprising a base medium and supplements, whereinthe low osmolality medium comprises: (a) a leukemia inhibitory factor(LIF) polypeptide; (b) a glycogen synthase kinase 3 (GSK3) inhibitor;and (c) a MEK inhibitor; wherein the base medium has an osmolality ofabout 180 mOsm/kg to about 250 mOsm/kg.

Further provided are methods for maintaining a population of hiPSCs inan in vitro culture, the methods comprising culturing the population ofhiPSCs in a low osmolality medium comprising a base medium andsupplements, wherein the low osmolality medium comprises: (a) a leukemiainhibitory factor (LIF) polypeptide; (b) a glycogen synthase kinase 3(GSK3) inhibitor; and (c) a MEK inhibitor; wherein the medium has anosmolality of about 175 mOsm/kg to about 280 mOsm/kg. Such methods canalso comprise culturing the population of hiPSCs in a low osmolalitymedium comprising a base medium and supplements, wherein the lowosmolality medium comprises: (a) a leukemia inhibitory factor (LIF)polypeptide; (b) a glycogen synthase kinase 3 (GSK3) inhibitor; and (c)a MEK inhibitor; wherein the base medium has an osmolality of about 180mOsm/kg to about 250 mOsm/kg.

In some methods, the hiPSCs comprise naïve or naïve-looking hiPSCs. Insome methods, the hiPSCs comprise naïve-like hiPSCs.

In some methods, the method enriches for a population of naïve ornaïve-looking hiPSCs. In some methods, the method enriches for apopulation of naïve-like hiPSCs.

In some methods, the transformed cells express reprogramming genescomprising Oct4, Sox2, Klf4, Myc, or any combination thereof. In somemethods, the transformed cells comprise primed hiPSCs.

In some methods, the base medium has an osmolality of about 200 mOsm/kg.In some methods, the base medium comprises NaCl at about 3 mg/ml, sodiumbicarbonate at about 2.2 mg/mL, and has an osmolality of about 200mOsm/kg.

In some methods, the base medium comprises glucose at about 4.5 mg/mL

In some methods, the low osmolality medium has an osmolality of about200 mOsm/kg to about 250 mOsm/kg. In some methods, the low osmolalitymedium has an osmolality of about 233 mOsm/kg.

In some methods, the supplements comprise: (a) F-12 medium; (b) N2supplement; (c) NEUROBASAL medium; (d) B-27 supplement; (e) L-glutamine;(f) 2-mercaptoethanol; or (g) any combination of (a) to (f).

In some methods, the LIF polypeptide is a human LIF (hLIF) polypeptide.In some methods, the GSK3 inhibitor comprises CHIR99021. In somemethods, the MEK inhibitor comprises PD0325901. In some methods, the lowosmolality medium comprises inhibitors consisting essentially of a GSK3inhibitor and a MEK inhibitor.

In some methods, the low osmolality medium comprises base medium atabout 24.75% (v/v), F-12 medium at about 24.75% (v/v), N2 supplement atabout 0.5% (v/v), NEUROBASAL medium at about 49% (v/v), B-27 supplementat about 1% (v/v), L-glutamine at about 2 mM, 2-mercaptoethanol at about0.1 mM, hLIF at about 100 units/mL, CHIR99021 at about 3 μM, andPD0325901 at about 0.5 μM.

In some methods, the low osmolality medium does not comprise one or moreof the following: bFGF supplement, TGF-β1 supplement, JNK inhibitor, p38inhibitor, ROCK inhibitor, and PKC inhibitor. In some methods, the lowosmolality medium does not comprise basic fibroblast growth factor(bFGF).

In some methods, the hiPSCs or the transformed cells are cultured onMATRIGEL™, newborn human foreskin fibroblast (NuFF) feeder cells, orGELTREX™.

In some methods, the hiPSCs express one or more pluripotency markers. Insome methods, the one or more pluripotency markers comprises NANOG,alkaline phosphatase, or a combination thereof. In some methods, thehiPSCs have a normal karyotype.

In some methods, the hiPSCs display a morphology characterized bycompact dome-shaped colonies.

In some methods, the hiPSCs can be enzymatically dissociated into asingle-cell suspension and subcultured. In some methods, the enzymaticdissociation is performed using trypsin. In some methods, the enzymaticdissociation can be performed in the absence of a Rho-associated proteinkinase (ROCK) inhibitor. In some methods, the subcultured hiPSCscontinue to express the one or more pluripotency markers. In somemethods, the subcultured hiPSCs maintain a naïve or naïve-looking stateand display a morphology characterized by compact dome-shaped colonies.In some methods, the subcultured hiPSCs maintain a normal karyotype.

In some methods, the hiPSCs can differentiate into cells of any one ofthe endoderm, ectoderm, or mesoderm germ layers.

In some methods, the hiPSCs have a doubling time of between about 16hours and about 24 hours.

In some methods, the transformed cells are first cultured in a highosmolality medium prior to culturing in the low osmolality medium,wherein the high osmolality medium comprises bFGF. Optionally, the highosmolality medium has an osmolality of at least about 290 mOsm/kg.

In some methods, the transformed cells are first cultured in the highosmolality medium until they express characteristics of a naïve ornaïve-looking state. In some methods, the transformed cells are firstcultured in the high osmolality medium for a period of about two months.In some methods, the transformed cells are first cultured in the highosmolality medium until they display a morphology characterized bythree-dimensional cell clumps.

Further provided are hiPSCs made by any of the above methods.

Further provided are methods for modifying a target genomic locus in ahiPSC, comprising: (a) introducing into the hiPSC a targeting vectorcomprising an insert nucleic acid flanked by 5′ and 3′ homology armscorresponding to 5′ and 3′ target sites at the target genomic locus; and(b) identifying a genetically modified hiPSC comprising in its genomethe insert nucleic acid integrated at the target genomic locus; whereinthe hiPSC is cultured in a low osmolality medium comprising a basemedium and supplements, wherein the low osmolality medium comprises: (a)a leukemia inhibitory factor (LIF) polypeptide; (b) a glycogen synthasekinase 3 (GSK3) inhibitor; and (c) a MEK inhibitor; wherein the mediumhas an osmolality of about 175 mOsm/kg to about 280 mOsm/kg. Suchmethods can also comprise: (a) introducing into the hiPSC a targetingvector comprising an insert nucleic acid flanked by 5′ and 3′ homologyarms corresponding to 5′ and 3′ target sites at the target genomiclocus; and (b) identifying a genetically modified hiPSC comprising inits genome the insert nucleic acid integrated at the target genomiclocus; wherein the hiPSC is cultured in a low osmolality mediumcomprising a base medium and supplements, wherein the low osmolalitymedium comprises: (a) a leukemia inhibitory factor (LIF) polypeptide;(b) a glycogen synthase kinase 3 (GSK3) inhibitor; and (c) a MEKinhibitor; wherein the base medium has an osmolality of about 180mOsm/kg to about 250 mOsm/kg. In some methods, the targeting vector is alarge targeting vector (LTVEC), wherein the sum total of the 5′ and 3′homology arms is at least 10 kb. In some methods, introducing step (a)further comprises introducing a nuclease agent that promotes homologousrecombination between the targeting vector and the target genomic locusin the hiPSC. In some methods, the targeted genetic modificationcomprises: (a) deletion of an endogenous human nucleic acid sequence;(b) insertion of an exogenous nucleic acid sequence; or (c) replacementof the endogenous human nucleic acid sequence with the exogenous nucleicacid sequence. In some methods, the exogenous nucleic acid sequencecomprises one or more of the following: (a) a nucleic acid sequence thatis homologous or orthologous to the endogenous human nucleic acidsequence; (b) a chimeric nucleic acid sequence; (c) a conditional alleleflanked by site-specific recombinase target sequences; and (d) areporter gene operably linked to a promoter active in the hiPSC.

Such methods for modifying a target genomic locus in a hiPSC, can alsocomprise: (a) introducing into the hiPSC one or more nuclease agentsthat induces one or more nicks or double-strand breaks at a recognitionsite at the target genomic locus; and (b) identifying at least one cellcomprising in its genome a modification at the target genomic locus;wherein the hiPSC is cultured in a low osmolality medium comprising abase medium and supplements, wherein the low osmolality mediumcomprises: (i) a leukemia inhibitory factor (LIF) polypeptide; (ii) aglycogen synthase kinase 3 (GSK3) inhibitor; and (iii) a MEK inhibitor;wherein the medium has an osmolality of about 175 mOsm/kg to about 280mOsm/kg. Such methods can also comprise: (a) introducing into the hiPSCone or more nuclease agents that induces one or more nicks ordouble-strand breaks at a recognition site at the target genomic locus;and (b) identifying at least one cell comprising in its genome amodification at the target genomic locus; wherein the hiPSC is culturedin a low osmolality medium comprising a base medium and supplements,wherein the low osmolality medium comprises: (i) a leukemia inhibitoryfactor (LIF) polypeptide; (ii) a glycogen synthase kinase 3 (GSK3)inhibitor; and (iii) a MEK inhibitor; wherein the base medium has anosmolality of about 180 mOsm/kg to about 250 mOsm/kg.

In any such methods for modifying a target genomic locus in a hiPSC, thehiPSCs can be enzymatically dissociated into a single-cell suspensionand subcultured prior to step (a). Optionally, the enzymaticdissociation is performed using trypsin. Optionally, the enzymaticdissociation is performed in the absence of a ROCK inhibitor. In somemethods, the subcultured hiPSCs continue to express one or morepluripotency markers. In some methods, the subcultured hiPSCs maintain anaïve or naïve-looking state and display a morphology characterized bycompact dome-shaped colonies. In some methods, the subcultured hiPSCsmaintain a normal karyotype.

In some methods, the nuclease agent comprises a zinc finger nuclease(ZFN). In some methods, the nuclease agent comprises a TranscriptionActivator-Like Effector Nuclease (TALEN). In some methods, the nucleaseagent comprises a Clustered Regularly Interspaced Short PalindromicRepeats (CRISPR) associated (Cas) protein and a guide RNA (gRNA)comprising a CRISPR RNA (crRNA) that recognizes a genomic targetsequence and a trans-activating CRISPR RNA (tracrRNA). Optionally, theCas protein is Cas9.

In some methods, the targeted genetic modification is biallelic.

In some methods, the hiPSCs comprise naïve or naïve-looking hiPSCs. Insome methods, the hiPSCs comprise naïve-like hiPSCs. In some methods,the hiPSCs express one or more pluripotency markers. Optionally, thepluripotency markers comprise NANOG, alkaline phosphatase, or acombination thereof. In some methods, the hiPSCs display a morphologycharacterized by compact dome-shaped colonies. In some methods, thehiPSCs can differentiate into cells of any one of the endoderm,ectoderm, or mesoderm germ layers. In some methods, the hiPSCs have adoubling time of between about 16 hours and about 24 hours. In somemethods, the hiPSCs have a normal karyotype.

In some methods, the hiPSCs are derived from non-pluripotent cellstransformed to express a pluripotent state. Optionally, the transformedcells express reprogramming genes comprising Oct4, Sox2, Klf4, Myc, orany combination thereof. Optionally, the transformed cells compriseprimed hiPSCs. In some methods, the transformed cells are first culturedin a high osmolality medium prior to culturing in the low osmolalitymedium, wherein the high osmolality medium comprises bFGF. Optionally,the high osmolality medium has an osmolality of at least 290 mOsm/kg. Insome methods, the transformed cells are first cultured in the highosmolality medium until they express characteristics of a naïve ornaïve-looking state. In some methods, the transformed cells are firstcultured in the high osmolality medium for a period of about two months.In some methods, the transformed cells are first cultured in the highosmolality medium until they display a morphology characterized bythree-dimensional cell clumps.

In some methods, the base medium has an osmolality of about 200 mOsm/kg.In some methods, the base medium comprises NaCl at about 3 mg/ml, sodiumbicarbonate at about 2.2 mg/mL, and has an osmolality of about 200mOsm/kg.

In some methods, the base medium comprises glucose at about 4.5 mg/mL

In some methods, the low osmolality medium has an osmolality of about200 mOsm/kg to about 250 mOsm/kg. In some methods, the low osmolalitymedium has an osmolality of about 233 mOsm/kg.

In some methods, the supplements comprise: (i) F-12 medium; (ii) N2supplement; (iii) NEUROBASAL medium; (iv) B-27 supplement; (v)L-glutamine; (vi) 2-mercaptoethanol; or (vii) any combination of (i) to(vi). In some methods, the LIF polypeptide is a human LIF (hLIF)polypeptide. In some methods, the GSK3 inhibitor comprises CHIR99021. Insome methods, the MEK inhibitor comprises PD0325901.

In some methods, the low osmolality medium comprises inhibitorsconsisting essentially of a glycogen synthase kinase 3 (GSK3) inhibitorand a MEK inhibitor.

In some methods, the low osmolality medium comprises base medium atabout 24.75% (v/v), F-12 medium at about 24.75% (v/v), N2 supplement atabout 0.5% (v/v), NEUROBASAL medium at about 49% (v/v), B-27 supplementat about 1% (v/v), L-glutamine at about 2 mM, 2-mercaptoethanol at about0.1 mM, hLIF at about 100 units/mL, CHIR99021 at about 3 μM, andPD0325901 at about 0.5 μM.

In some methods, the low osmolality medium does not comprise one or moreof the following: bFGF supplement; TGF-β1 supplement; JNK inhibitor; p38inhibitor; ROCK inhibitor; and PKC inhibitor. In some methods, the lowosmolality medium does not comprise bFGF supplement.

In some methods, the hiPSCs are cultured on MATRIGEL, NuFF feeder cells,or GELTREX.

Further provided are modified hiPSCs made by any of the above methods.

Further provided are in vitro cultures comprising: (a) a population ofhiPSCs; and (b) a low osmolality medium comprising a base medium andsupplements, wherein the low osmolality medium comprises: (i) a leukemiainhibitory factor (LIF) polypeptide; (ii) a glycogen synthase kinase 3(GSK3) inhibitor; and (iii) a MEK inhibitor; wherein the medium has anosmolality of about 175 mOsm/kg to about 280 mOsm/kg. Such in vitrocultures can also comprise (a) a population of hiPSCs; and (b) a lowosmolality medium comprising a base medium and supplements, wherein thelow osmolality medium comprises: (i) a leukemia inhibitory factor (LIF)polypeptide; (ii) a glycogen synthase kinase 3 (GSK3) inhibitor; and(iii) a MEK inhibitor; wherein the base medium has an osmolality ofabout 180 mOsm/kg to about 250 mOsm/kg.

Further provided are populations of hiPSCs made or maintained in a lowosmolality medium comprising a base medium and supplements, wherein thelow osmolality medium comprises: (a) a leukemia inhibitory factor (LIF)polypeptide; (b) a glycogen synthase kinase 3 (GSK3) inhibitor; and (c)a MEK inhibitor; wherein the medium has an osmolality of about 175mOsm/kg to about 280 mOsm/kg. Such populations of hiPSCs can also bemade or maintained in a low osmolality medium comprising a base mediumand supplements, wherein the low osmolality medium comprises: (a) aleukemia inhibitory factor (LIF) polypeptide; (b) a glycogen synthasekinase 3 (GSK3) inhibitor; and (c) a MEK inhibitor; wherein the basemedium has an osmolality of about 180 mOsm/kg to about 250 mOsm/kg.

In some populations or in vitro cultures, the hiPSCs comprise naïve ornaïve-looking hiPSCs. In some populations or in vitro cultures, thehiPSCs comprise naïve-like hiPSCs.

In some populations or in vitro cultures, the hiPSCs are derived fromnon-pluripotent cells transformed to express a pluripotent state. Insome populations or in vitro cultures, the transformed cells expressreprogramming genes comprising Oct4, Sox2, Klf4, Myc, or any combinationthereof. In some populations or in vitro cultures, the transformed cellscomprise primed hiPSCs.

In some populations or in vitro cultures, the base medium has anosmolality of about 200 mOsm/kg. In some populations or in vitrocultures, the base medium comprises NaCl at about 3 mg/ml, sodiumbicarbonate at about 2.2 mg/mL, and has an osmolality of about 200mOsm/kg.

In some populations or in vitro cultures, the base medium comprisesglucose at about 4.5 mg/mL.

In some populations or in vitro cultures, the low osmolality mediumcomprising the base medium and supplements has an osmolality of about200 mOsm/kg to about 250 mOsm/kg. In some populations or in vitrocultures, the low osmolality medium has an osmolality of about 233mOsm/kg.

In some populations or in vitro cultures, the supplements comprise: (a)F-12 medium; (b) N2 supplement; (c) NEUROBASAL medium; (d) B-27supplement; (e) L-glutamine; (f) 2-mercaptoethanol; or (g) anycombination of (a) to (f).

In some populations or in vitro cultures, the LIF polypeptide is a humanLIF (hLIF) polypeptide. In some populations or in vitro cultures, theGSK3 inhibitor comprises CHIR99021. In some populations or in vitrocultures, the MEK inhibitor comprises PD0325901. In some populations orin vitro cultures, the low osmolality medium comprises inhibitorsconsisting essentially of a GSK3 inhibitor and a MEK inhibitor.

In some populations or in vitro cultures, the low osmolality mediumcomprises base medium at about 24.75% (v/v), F-12 medium at about 24.75%(v/v), N2 supplement at about 0.5% (v/v), NEUROBASAL medium at about 49%(v/v), B-27 supplement at about 1% (v/v), L-glutamine at about 2 mM,2-mercaptoethanol at about 0.1 mM, hLIF at about 100 units/mL, CHIR99021at about 3 μM, and PD0325901 at about 0.5 μM.

In some populations or in vitro cultures, the low osmolality medium doesnot comprise one or more of the following: bFGF supplement, TGF-β1supplement, JNK inhibitor, p38 inhibitor, ROCK inhibitor, and PKCinhibitor. In some populations or in vitro cultures, the low osmolalitymedium does not comprise basic fibroblast growth factor (bFGF).

In some populations or in vitro cultures, the hiPSCs or the transformedcells are cultured on MATRIGEL™, newborn human foreskin fibroblast(NuFF) feeder cells, or GELTREX™.

In some populations or in vitro cultures, the hiPSCs express one or morepluripotency markers. In some populations or in vitro cultures, the oneor more pluripotency markers comprises NANOG, alkaline phosphatase, or acombination thereof. In some populations or in vitro cultures, thehiPSCs have a normal karyotype.

In some populations or in vitro cultures, the hiPSCs display amorphology characterized by compact dome-shaped colonies.

In some populations or in vitro cultures, the hiPSCs can beenzymatically dissociated into a single-cell suspension and subcultured.In some populations or in vitro cultures, the enzymatic dissociation isperformed using trypsin. In some populations or in vitro cultures, theenzymatic dissociation can be performed in the absence of aRho-associated protein kinase (ROCK) inhibitor. In some populations orin vitro cultures, the subcultured hiPSCs continue to express the one ormore pluripotency markers. In some populations or in vitro cultures, thesubcultured hiPSCs maintain a naïve or naïve-looking state and display amorphology characterized by compact dome-shaped colonies. In somepopulations or in vitro cultures, the subcultured hiPSCs maintain anormal karyotype.

In some populations or in vitro cultures, the hiPSCs can differentiateinto cells of any one of the endoderm, ectoderm, or mesoderm germlayers.

In some populations or in vitro cultures, the hiPSCs have a doublingtime of between about 16 hours and about 24 hours.

In some populations or in vitro cultures, the transformed cells arefirst cultured in a high osmolality medium prior to culturing in the lowosmolality medium, wherein the high osmolality medium comprises bFGF.Optionally, the high osmolality medium has an osmolality of at leastabout 290 mOsm/kg.

In some populations or in vitro cultures, the transformed cells arefirst cultured in the high osmolality medium until they expresscharacteristics of a naïve or naïve-looking state. In some populationsor in vitro cultures, the transformed cells are first cultured in thehigh osmolality medium for a period of about two months. In somepopulations or in vitro cultures, the transformed cells are firstcultured in the high osmolality medium until they display a morphologycharacterized by three-dimensional cell clumps.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic for replacement of a portion of the humanADAM6 locus with a nucleic acid comprising the mouse Adam6a and mouseAdam6b loci using an LTVEC and a guide RNA in human iPS cells. Thetarget site for the guide RNA is indicated by the arrow.

FIG. 2A depicts the morphology displayed by human iPS cells cultured for8 days in 2i medium.

FIG. 2B depicts the morphology displayed by human iPS cells cultured for12 days in 2i medium.

FIGS. 3A-3D depict the morphology of human iPS cells cultured inmTeSR™-hLIF medium or low osmolality VG2i medium for 6 days. FIGS. 3Aand 3B depict the morphology of human iPS cells cultured in mTeSR™-hLIFmedium (FIG. 3A) or VG2i medium (FIG. 3B) for 6 days. FIGS. 3C and 3Ddepict the morphology of human iPS cells cultured on newborn humanforeskin fibroblast (NuFF) feeder cells in mTeSR™-hLIF medium (FIG. 3C)or VG2i medium (FIG. 3D) for 6 days.

FIG. 4A depicts reprogrammed human iPS cells cultured in VG2i mediumthat have been stained for alkaline phosphatase. FIGS. 4B and 4C depictreprogrammed human iPS cells cultured in VG2i medium that have beenimmunostained for the expression of NANOG.

FIGS. 5A-5C illustrate enzymatic dissociation and subculture ofreprogrammed human iPS cells cultured in VG2i medium. FIG. 5A depictsreprogrammed human iPS cells cultured in VG2i medium prior to enzymaticdissociation with trypsin in the absence of a ROCK inhibitor. FIG. 5Bdepicts human iPS cells cultured in VG2i medium for 1 day aftersubculture. FIG. 5C depicts human iPS cells cultured in VG2i medium for4 days after subculture.

FIGS. 6A and 6B depict the karyotypes of cells from two different humaniPS cell clones at passage 10 following dissociation with trypsin tocreate a single-cell suspension.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 sets forth a nucleic acid sequence comprised by ADAM6 gRNA.

SEQ ID NO: 2 sets forth the nucleic acid sequence of a target sequencefor a CRISPR/Cas complex.

Definitions

The terms “protein,” “polypeptide,” and “peptide,” used interchangeablyherein, include polymeric forms of amino acids of any length, includingcoded and non-coded amino acids and chemically or biochemically modifiedor derivatized amino acids. The terms also include polymers that havebeen modified, such as polypeptides having modified peptide backbones.

The terms “nucleic acid” and “polynucleotide,” used interchangeablyherein, include polymeric forms of nucleotides of any length, includingribonucleotides, deoxyribonucleotides, or analogs or modified versionsthereof. They include single-, double-, and multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purinebases, pyrimidine bases, or other natural, chemically modified,biochemically modified, non-natural, or derivatized nucleotide bases.

“Codon optimization” generally includes a process of modifying a nucleicacid sequence for enhanced expression in particular host cells byreplacing at least one codon of the native sequence with a codon that ismore frequently or most frequently used in the genes of the host cellwhile maintaining the native amino acid sequence. For example, a nucleicacid encoding a Cas protein can be modified to substitute codons havinga higher frequency of usage in a human cell. Codon usage tables arereadily available, for example, at the “Codon Usage Database.” Thesetables can be adapted in a number of ways. See Nakamura et al. (2000)Nucleic Acids Research 28:292. Computer algorithms for codonoptimization of a particular sequence for expression in a particularhost are also available (see, e.g., Gene Forge).

“Operable linkage” or being “operably linked” includes juxtaposition oftwo or more components (e.g., a promoter and another sequence element)such that both components function normally and allow the possibilitythat at least one of the components can mediate a function that isexerted upon at least one of the other components. For example, apromoter can be operably linked to a coding sequence if the promotercontrols the level of transcription of the coding sequence in responseto the presence or absence of one or more transcriptional regulatoryfactors.

“Complementarity” of nucleic acids means that a nucleotide sequence inone strand of nucleic acid, due to orientation of its nucleobase groups,forms hydrogen bonds with another sequence on an opposing nucleic acidstrand. The complementary bases in DNA are typically A with T and C withG. In RNA, they are typically C with G and U with A. Complementarity canbe perfect or substantial/sufficient. Perfect complementarity betweentwo nucleic acids means that the two nucleic acids can form a duplex inwhich every base in the duplex is bonded to a complementary base byWatson-Crick pairing. “Substantial” or “sufficient” complementary meansthat a sequence in one strand is not completely and/or perfectlycomplementary to a sequence in an opposing strand, but that sufficientbonding occurs between bases on the two strands to form a stable hybridcomplex in set of hybridization conditions (e.g., salt concentration andtemperature). Such conditions can be predicted by using the sequencesand standard mathematical calculations to predict the Tm of hybridizedstrands, or by empirical determination of Tm by using routine methods.Tm includes the temperature at which a population of hybridizationcomplexes formed between two nucleic acid strands are 50% denatured. Ata temperature below the Tm, formation of a hybridization complex isfavored, whereas at a temperature above the Tm, melting or separation ofthe strands in the hybridization complex is favored. Tm may be estimatedfor a nucleic acid having a known G+C content in an aqueous 1 M NaClsolution by using, e.g., Tm=81.5+0.41(% G+C), although other known Tmcomputations take into account nucleic acid structural characteristics.

“Hybridization condition” includes the cumulative environment in whichone nucleic acid strand bonds to a second nucleic acid strand bycomplementary strand interactions and hydrogen bonding to produce ahybridization complex. Such conditions include the chemical componentsand their concentrations (e.g., salts, chelating agents, formamide) ofan aqueous or organic solution containing the nucleic acids, and thetemperature of the mixture. Other factors, such as the length ofincubation time or reaction chamber dimensions may contribute to theenvironment. See, e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2.sup.nd ed., pp. 1.90-1.91, 9.47-9.51, 1 1.47-11.57 (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Hybridization requires that the two nucleic acids contain complementarysequences, although mismatches between bases are possible. Theconditions appropriate for hybridization between two nucleic acidsdepend on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof complementation between two nucleotide sequences, the greater thevalue of the melting temperature (Tm) for hybrids of nucleic acidshaving those sequences. For hybridizations between nucleic acids withshort stretches of complementarity (e.g., complementarity over 35 orfewer, 30 or fewer, 25 or fewer, 22 or fewer, 20 or fewer, or 18 orfewer nucleotides) the position of mismatches becomes important (seeSambrook et al., supra, 11.7-11.8). Typically, the length for ahybridizable nucleic acid is at least about 10 nucleotides. Illustrativeminimum lengths for a hybridizable nucleic acid include at least about15 nucleotides, at least about 20 nucleotides, at least about 22nucleotides, at least about 25 nucleotides, and at least about 30nucleotides. Furthermore, the temperature and wash solution saltconcentration may be adjusted as necessary according to factors such aslength of the region of complementation and the degree ofcomplementation.

The sequence of polynucleotide need not be 100% complementary to that ofits target nucleic acid to be specifically hybridizable. Moreover, apolynucleotide may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridizationevent (e.g., a loop structure or hairpin structure). A polynucleotide(e.g., gRNA) can comprise at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or 100% sequence complementarity to a targetregion within the target nucleic acid sequence to which they aretargeted. For example, a gRNA in which 18 of 20 nucleotides arecomplementary to a target region, and would therefore specificallyhybridize, would represent 90% complementarity. In this example, theremaining noncomplementary nucleotides may be clustered or interspersedwith complementary nucleotides and need not be contiguous to each otheror to complementary nucleotides.

Percent complementarity between particular stretches of nucleic acidsequences within nucleic acids can be determined routinely using BLASTprograms (basic local alignment search tools) and PowerBLAST programsknown in the art (Altschul et al. (1990) J. Mol. Biol. 215:403-410;Zhang and Madden (1997) Genome Res. 7:649-656) or by using the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, Madison Wis.), usingdefault settings, which uses the algorithm of Smith and Waterman (Adv.Appl. Math., 1981, 2, 482-489).

“Sequence identity” or “identity” in the context of two polynucleotidesor polypeptide sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window. When percentage of sequence identity isused in reference to proteins it is recognized that residue positionswhich are not identical often differ by conservative amino acidsubstitutions, where amino acid residues are substituted for other aminoacid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. When sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically, this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

“Percentage of sequence identity” includes the value determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison, andmultiplying the result by 100 to yield the percentage of sequenceidentity.

Unless otherwise stated, sequence identity/similarity values include thevalue obtained using GAP Version 10 using the following parameters: %identity and % similarity for a nucleotide sequence using GAP Weight of50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; %identity and % similarity for an amino acid sequence using GAP Weight of8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or anyequivalent program thereof. “Equivalent program” includes any sequencecomparison program that, for any two sequences in question, generates analignment having identical nucleotide or amino acid residue matches andan identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” a protein maycontain the protein alone or in combination with other ingredients.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

The term “about” means that the specified value can vary by somepercentage. In some examples, the percentage can be 1, 2, 3, 4, 8, or10% of the specified value.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a cell” or “at least one cell” can include a plurality ofcells, including mixtures thereof.

DETAILED DESCRIPTION A. Low Osmolality Medium for Making and MaintainingHuman Induced Pluripotent Stem Cells.

A cell culture medium is provided for use in the methods andcompositions of the invention. In one embodiment, the medium is suitablefor making a population of human iPS cells. In another embodiment, themedium is suitable for maintaining human iPS cells in culture. In someembodiments, the human iPS cells are naïve or naïve-looking.

The medium provided herein comprises at least a base medium,supplements, a leukemia inhibitory factor (LIF) polypeptide, a glycogensynthase kinase 3 (GSK3) inhibitor, and a mitogen-activated proteinkinase kinase (MEK) inhibitor. A “base medium” or “base media” includes,for example, a base medium known in the art (e.g., Dulbecco's ModifiedEagle's Medium (DMEM)) that is suitable for use (with added supplements)in growing or maintaining pluripotent cells (e.g., iPS cells) inculture. Base medium is typically supplemented with a number ofsupplements known in the art when used to maintain cells in culture.

The present medium is a low osmolality medium. In one example, theosmolality is between about 175-280 mOsm/kg. In further examples, theosmolality of the medium is about 180-270 mOsm/kg, about 200-250mOsm/kg, about 220-240 mOsm/kg, or about 225-235 mOsm. In a particularembodiment, the osmolality of the medium is about 233 mOsm/kg.

The base medium provided for the invention is a low osmolality basemedium to which supplements are added. The present base medium differsfrom base media typically used to maintain human iPS cells in culture,which include Dulbecco's Modified Eagle's Medium (DMEM), in variousforms (e.g., Invitrogen DMEM, Cat. No. 1 1971 -025), and a low salt DMEMavailable commercially as KO-DMEM™ (Invitrogen Cat. No. 10829-018).

The base medium provided herein is a low osmolality medium but exhibitscharacteristics that are not limited to low osmolality. For example, theDMEM formulation shown in Table 1 can be made suitable for the purposesof the invention by altering the sodium chloride and/or sodiumbicarbonate concentrations as provided herein, which will result in adifferent osmolality as compared with the standard DMEM base medium orlow-salt DMEM base medium (KO-DMEM) shown in Table 1.

TABLE 1 DMEM base medium formulation. Component Mg/L mM Glycine 30 0.4L-Arginine•HCl 84 0.398 L-Cystine•2HCl 63 0.201 L-Glutamine 584 4L-Histidine•HCl•H2O 42 0.2 L-Isoleucine 105 0.802 L-Leucine 105 0.802L-Lysine•HCl 146 0.798 L-Methionine 30 0.201 L-Phenylalanine 66 0.4L-Serine 42 0.4 L-Threonine 95 0.798 L-Tryptophan 16 0.0784 L-Tyrosinedisodium salt dihydrate 104 0.398 L-Valine 94 0.803 Choline chloride 40.0286 D-Calcium pantothenate 4 8.39 × 10⁻³ Folic Acid 4 9.07 × 10⁻³Niacinamide 4 0.0328 Pyridoxine•HCl 4 0.0196 Riboflavin 0.4 1.06 × 10⁻³Thiamine•HCl 4 0.0119 i-Inositol 7.2 0.04 Calcium Chloride (CaCl₂)(anhydrous) 200 1.8 Ferric Nitrate (Fe(NO₃)₃•9H₂O) 0.1 2.48 × 10⁻⁴Magnesium Sulfate (MgSO₄) (anhyd.) 97.67 0.814 Potassium Chloride (KCl)400 5.33 D-Glucose (Dextrose) 4500 25 Phenol Red 15 0.0399 NaCl/NaHCO₃Content of DMEM Sodium Bicarbonate (NaHCO₃) 3700 44.05 Sodium Chloride(NaCl) 6400 110.34 Osmolality 340 mOsm/kg NaCl/NaHCO₃ Content of LowSalt DMEM (KO-DMEM) Sodium Bicarbonate (NaHCO₃) 2200 26 Sodium Chloride(NaCl) 5100 87.7 Osmolality 275 mOsm/kg NaCl/NaHCO₃ Content of LowOsmolality DMEM (VG-DMEM) Sodium Bicarbonate (NaHCO₃) 2200 26 SodiumChloride (NaCl) 3000 50 Osmolality 200 mOsm/kg

The present base medium can include a salt of an alkaline metal and ahalide, such as sodium chloride (NaCl). Exemplary concentrations of NaClin the base medium include 50±5 mM or about 3 mg/mL. The concentrationof a salt of an alkaline metal and a halide in the base medium or amedium comprising the base medium and supplements can be, for example,no more than about 100, 90, 80, 70, 60, or 50 mM. For example, the basemedium or a medium comprising the base medium and supplements cancomprise a concentration of a salt of an alkaline metal and halide ofabout 50-110, 60-105, 70-95, 80-90, 90 mM, or 85 mM. Alternatively, theconcentration of a salt of an alkaline metal and halide can be, forexample, 50±5 mM, 87±5 mM, 110±5 mM, about 3 mg/mL, about 5.1 mg/mL, orabout 6.4 mg/mL.

In another embodiment, the base medium exhibits a concentration of asalt of carbonic acid. The salt of carbonic acid can be a sodium salt.In such an example, the sodium salt can be sodium bicarbonate. In aparticular embodiment, sodium bicarbonate is present in the base mediumat a concentration of about 26±5 mM or about 2.2 mg/mL. Theconcentration of a salt of carbonic acid in the base medium or a mediumcomprising the base medium and supplements can be, for example, no morethan 45, 40, 35, 30, 25, or 20 mM. For example, the base medium or amedium comprising the base medium and supplements can comprise aconcentration of carbonic acid salt in the base medium of about 10-40,18-44, 17-30, 18-26, 13-25, 20-30, 25-26, 18, or 26 mM. Alternatively,the concentration of carbonic acid salt can be, for example, 18±5 mM,26±5 mM, about 1.5 mg/mL, or about 2.2 mg/mL.

The sum of the concentration of the salt of the alkaline metal andhalide and the salt of carbonic acid in the base medium or a mediumcomprising the base medium and supplements can be, for example, no morethan 140, 130, 120, 110, 100, 90, or 80 mM. For example, the base mediumor a medium comprising the base medium and supplements can comprise asum concentration of a salt of an alkaline metal and halide and a saltof carbonic acid of about 80-140, 85-130, 90-120, 95-120, 100-120, or115 mM.

The molar ratio of the salt of the alkaline metal and halide and thesalt of carbonic acid in the base medium or a medium comprising the basemedium and supplements can be, for example, higher than 2.5. Forexample, the base medium or a medium comprising the base medium andsupplements can comprise a molar ratio of a salt of an alkaline metaland halide and a salt of carbonic acid of about 2.6-4.0, 2.8-3.8,3.0-3.6, 3.2-3.4, 3.3-3.5, or 3.4.

In yet another embodiment, the base medium is a low osmolality basemedium. The osmolality of the base medium can be within a range of about175-280 mOsm/kg, about 180-250 mOsm/kg, about 190-225 mOsm/kg, or about195-205 mOsm/kg. An exemplary osmolality of the base medium can be 200,214, 216, or 218 mOsm/kg. In a particular example, the osmolality of thebase medium is 200 mOsm/kg. The osmolality can be determined when cellsare cultured in different concentrations of CO2. In some examples, cellsare cultured at 3% CO2 or 5% CO2. The osmolality of the base medium or amedium comprising the base medium and supplements can be, for example,no more than about 330, 320, 310, 300, 290, 280, 275, 270, 260, 250,240, 230, 220, 210, or 200 mOsm/kg. For example, the base medium or themedium comprising the base medium and supplements can comprise anosmolality of about 200-329, 218-322, 240-320, 250-310, 275-295, or260-300 mOsm/kg. For example, the base medium or the medium comprisingthe base medium and the supplements can comprise an osmolality of about270 mOsm/kg, about 261 mOsm/kg, or about 218 mOsm/kg. Alternatively, theosmolality can be 218±22 mOsm/kg, 261±26 mOsm/kg, 294±29 mOsm/kg, or322±32 mOsm/kg.

The osmolality of the base medium can be, for example, about 130-270,140-260, 150-250, 160-240, 170-230, 180-220, 190-210, 195-205, or 200mOsm/kg. Alternatively, the osmolality of the base medium can be, forexample, about 200±70, 200±60, 200±50, 200 ±40, 200 ±35, 200±30, 200±25,200±20, 200±15, 200±10, 200±5, or 200 mOsm/kg. Alternatively, theosmolality of the base medium can be, for example, about 130-140, about140-150, about 150-160, about 160-170, about 170-180, about 180-190,about 190-200, about 200-210, about 210-220, about 220-230, about230-240, about 240-250, about 250-260, about 260-270, about 270-280,about 280-290, about 290-300, about 300-310, about 310-320, or about320-330 mOsm/kg. Alternatively, the osmolality of the base medium canbe, for example, less than about 330, 320, 310, 300, 290, 280, 270, 260,250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, or 130mOsm/kg.

The osmolality of the medium comprising the base medium and supplementscan be, for example, about 205-260, 215-250, 225-240, 230-235, or 233mOsm/kg. Alternatively, the osmolality of the medium comprising the basemedium and supplements can be, for example, about 233±27, 233±25,233±20, 233±15, 233±10, 233±5, or 233 mOsm/kg. Alternatively, theosmolality of the medium comprising the base medium and supplements canbe, for example, about 200-205, 205-210, 210-215, 215-220, 220-225,225-230, 230-235, 235-240, 240-245, 245-250, 250-255, or 255-260mOsm/kg. Alternatively, the osmolality of the medium comprising the basemedium and supplements can be, for example, less than 260, 255, 250,245, 240, 235, 230, 225, 220, 215, 210, 205, or 200 mOsm/kg.

In some low osmolality media, the base medium comprises about 87±5 mMNaCl and about 26±5 mM carbonate. For example the base media cancomprise about 5.1 mg/mL NaCl, about 2.2 mg/mL sodium bicarbonate, andan osmolality of about 275 mOsm/kg.

In some low osmolality media, the base medium comprises about 50±5 mMNaCl and about 26±5 mM carbonate. For example, the base medium cancomprise about 3.0 mg/mL NaCl, about 2.2 mg/mL sodium bicarbonate, andan osmolality of about 200 mOsm/kg. In a preferred embodiment, the basemedium comprises NaCl at a concentration of 3.0 mg/mL, sodiumbicarbonate at a concentration of about 2.2 mg/mL, and has an osmolalityof 200 mOsm/kg.

Other examples of low osmolality media are described in WO 2011/156723,US 2011/0307968, and US 2015/0067901, each of which is hereinincorporated by reference in its entirety.

Supplements formulated with the base medium of the invention aresuitable for making, maintaining, or enriching populations of human iPScells disclosed herein. Such supplements are indicated as “supplements”or “+supplements” in this disclosure. The term “supplements” or thephrase “+supplements,” includes one or more additional elements added tothe components of the base medium described in Table 1. For example,supplements can include, without limitation, F-12® medium (Gibco), N2®supplement (Gibco; 100X solution), NEUROBASAL® medium (Gibco), B-27®supplement (Gibco; 50X solution), L-glutamine, glucose,2-mercaptoethanol, a Leukemia Inhibitory Factor (LIF) polypeptide, aglycogen synthase kinase 3 inhibitor, a MEK inhibitor, or anycombination thereof. Supplements can also include, for example, fetalbovine serum (FBS), antibiotic(s), penicillin and streptomycin (i.e.,penstrep), pyruvate salts (e.g., sodium pyruvate), and nonessentialamino acids (e.g., MEM NEAA).

In a particular embodiment, the LIF polypeptide is a human LIF (hLIF)polypeptide. In some examples, a hLIF polypeptide is used at aconcentration of about 1-1000 units/mL, about 20-800 units/mL, about50-500 units/mL, about 75-250 units/mL, or about 100 units/mL.

The media can comprise inhibitors, for example, consisting essentiallyof a GSK3 inhibitor and a MEK inhibitor. For example, the medium cancomprise inhibitors consisting of a GSK3 inhibitor and a MEK inhibitor.

In another particular embodiment, the GSK3 inhibitor comprisesCHIR99021. In some examples, CHIR99021 is used at a concentration ofabout 0.1 to 10 04, about 1-5 04, about 2-4 μM, or about 3 μM4.

In another particular embodiment, the MEK inhibitor comprises PD0325901.In some examples, PD0325901 is used at a concentration of about 0.1-5μM, about 0.2-1 μM, about 0.3-0.7 μM, or about 0.5 μM.

An exemplary medium comprises a low osmolality base medium describedherein at about 24.75% (v/v), F-12 medium at about 24.75% (v/v), N2supplement at about 0.5% (v/v), NEUROBASAL medium at about 49% (v/v),B-27 supplement at about 1% (v/v), L-glutamine at about 2 mM,2-mercaptoethanol at about 0.1 mM, hLIF at about 100 units/mL, CHIR99021at about 3 μM, and PD0325901 at about 0.5 04.

In another particular embodiment, the medium may or may not comprisebasic fibroblast growth factor (bFGF, also known as FGF2 or FGF-β1).Preferably the present medium does not comprise bFGF.

The medium may or may not comprise one or more of transforming growthfactor beta 1 (TGF-β1) supplement, bFGF supplement, c-Jun N-terminalkinase (INK) inhibitor (e.g., SP600125), p38 mitogen-activated proteinkinase (p38) inhibitor (e.g., SB203580), rho-associated protein kinase(ROCK) inhibitor (e.g., Y-27632), and protein kinase C (PKC) inhibitor(e.g., Go6983). The medium may or may not comprise forskolin. Forexample, some media do not comprise one or more of TGF-β1 supplement,bFGF supplement, JNK inhibitor (e.g., SP600125), p38 inhibitor (e.g.,SB203580), ROCK inhibitor (e.g., Y-27632), and PKC inhibitor (e.g.,Go6983). Some media do not comprise one or more of p38 inhibitor and JNKinhibitor. Some media do not comprise bFGF supplement or TGF-β1supplement. Some media do not comprise TGF-β1 supplement. Some media donot comprise any one of TGF-β1 supplement, bFGF supplement, JNKinhibitor (e.g., SP600125), p38 inhibitor (e.g., SB203580), ROCKinhibitor (e.g., Y-27632), and PKC inhibitor (e.g., Go6983). Some mediado not comprise forskolin.

B. Human Induced Pluripotent Stem Cells

Methods and compositions are provided herein for making a population ofhuman iPS cells. Methods and compositions are further provided formaintaining human iPS cells in culture. Human iPS cells that areproduced or maintained in culture are also provided.

The term “pluripotent cell” or “pluripotent stem cell” includes anundifferentiated cell that possesses the ability to develop into morethan one differentiated cell type. Such pluripotent cells can be, forexample, a mammalian embryonic stem (ES cell) cell or a mammalianinduced pluripotent stem cell (iPS cell). Examples of pluripotent cellsinclude human iPS cells.

The term “embryonic stem cell” or “ES cell” means an embryo-derivedtotipotent or pluripotent stem cell, derived from the inner cell mass ofa blastocyst, that can be maintained in an in vitro culture undersuitable conditions. ES cells are capable of differentiating into cellsof any of the three vertebrate germ layers, e.g., the endoderm, theectoderm, or the mesoderm. ES cells are also characterized by theirability propagate indefinitely under suitable in vitro cultureconditions. See, for example, Thomson et al. (Science (1998) Vol.282(5391), pp. 1145-1147).

The term “induced pluripotent stem cell” or “iPS cell” includes apluripotent stem cell that can be derived directly from a differentiatedadult cell. Human iPS cells can be generated by introducing specificsets of reprogramming factors into a non-pluripotent cell which caninclude, for example, Oct3/4, Sox family transcription factors (e.g.,Soxl, Sox2, Sox3, Sox15), Myc family transcription factors (e.g., c-Myc,1-Myc, n-Myc), Krüppel-like family (KLF) transcription factors (e.g.,KLF1, KLF2, KLF4, KLF5), and/or related transcription factors, such asNANOG, LIN28, and/or Glisl. Human iPS cells can also be generated, forexample, by the use of miRNAs, small molecules that mimic the actions oftranscription factors, or lineage specifiers. Human iPS cells arecharacterized by their ability to differentiate into any cell of thethree vertebrate germ layers, e.g., the endoderm, the ectoderm, or themesoderm. Human iPS cells are also characterized by their abilitypropagate indefinitely under suitable in vitro culture conditions. See,for example, Takahashi and Yamanaka (Cell (2006) Vol. 126(4), pp.663-676).

The terms “naïve” and “primed” identify different pluripotency states ofhuman iPS cells. The term “naïve-looking” identifies a cell expressing apluripotent state that exhibits one or more characteristics of a naïvepluripotent cell. Naïve-looking human iPS cells can also be referred toas “naïve-like” human iPS cells. The terms “naïve-looking” and“naïve-like” are intended to be equivalent. In some embodiments,naïve-looking human iPS cells exhibit one or more morphologicalcharacteristics of naïve human iPS cells, such as a morphologycharacterized by compact dome-shaped colonies. In some embodiments,naïve-looking human iPS cells express one or more of the pluripotencymarkers described herein. In some embodiments, naïve or naïve-lookinghuman iPS cells are naïve human iPS cells. In other embodiments, naïveor naïve-looking human iPS cells are naïve-looking iPS cells.

Characteristics of naïve and primed iPS cells are described in the art.See, for example, Nichols and Smith (Cell Stem Cell (2009) Vol. 4(6),pp. 487-492). Naïve human iPS cells exhibit a pluripotency state similarto that of ES cells of the inner cell mass of a pre-implantation embryo.Such naïve cells are not primed for lineage specification andcommitment. Female naïve iPS cells are characterized by two active Xchromosomes. In culture, self-renewal of naïve human iPS cells isdependent on leukemia inhibitory factor (LIF) and other inhibitors.Cultured naïve human iPS cells display a clonal morphology characterizedby rounded dome-shaped colonies and a lack of apico-basal polarity.Cultured naïve cells can further display one or more pluripotency makersas described elsewhere herein. Under appropriate conditions, thedoubling time of naïve human iPS cells in culture can be between 16 and24 hours.

Primed human iPS cells express a pluripotency state similar to that ofpost-implantation epiblast cells. Such cells are primed for lineagespecification and commitment. Female primed iPS cells are characterizedby one active X chromosome and one inactive X chromosome. In culture,self-renewal of primed human iPS cells is dependent on fibroblast growthfactor (FGF) and activin. Cultured primed human iPS cells display aclonal morphology characterized by an epithelial monolayer and displayapico-basal polarity. Under appropriate conditions, the doubling time ofprimed human iPS cells in culture can be 24 hours or more.

In one embodiment, human iPS cells can be derived from non-pluripotentcells transformed to express a pluripotent state. Such transformed cellsinclude, for example, cells that have been transformed to expressreprogramming genes that induce pluripotency. A pluripotent state caninclude, for example, expression of one or more of the pluripotencymarkers described herein. Such cells (such as human foreskinfibroblasts) can be transformed to express reprogramming genes, or anyadditional genes of interest, by any means known in the art. See, forexample, Takahashi and Yamanaka (Cell (2006) Vol. 126(4), pp. 663-676).For example, they can be introduced into the cells using one or moreplasmids, lentiviral vectors, or retroviral vectors. In some cases, thevectors integrate into the genome and can be removed after reprogrammingis complete. In particular embodiments, the non-pluripotent cells aretransformed with reprogramming genes comprising Oct4, Sox2, Klf4, Myc,or any combination thereof. In some examples, the transformed cellscomprise primed human iPS cells.

In some embodiments, the human iPS cells cultured in the low osmolalitymedium described herein express one or more phenotypes, gene expressionprofiles, or markers characteristic of a naïve state. In one example,the human iPS cells express one or more pluripotency markers whoseexpression is indicative of a naïve state. Such pluripotency markers caninclude alkaline phosphatase, NANOG, 5T4, ABCG2, Activin RIB/ALK-4,Activin RIIB, E-Cadherin, Cbx2, CD9, CD30/TNFRSF8, CD117/c-kit, CDX2,CHD1, Cripto, DNMT3B, DPPA2, DPPA4, DPPA5/ESG1, EpCAM/TROP1, ERRbeta/NR3B2, ESGP, F-box protein 15/FBXO15, FGF-4, FGF-5, FoxD3, GBX2,GCNF/NR6A1, GDF-3, Gi24/VISTA/B7-H5, integrin alpha 6/CD49f, integrinalpha 6 beta 1, integrin alpha 6 beta 4, integrin beta 1/CD29, KLF4,KLF5, L1TD1, Lefty, Lefty-1, Lefty-A, LIN-28A, LIN-28B, LIN-41, cMaf,cMyc, Oct-3/4, Oct-4A, Podocalyxin, Rex-1/ZFP42, Smad2, Smad2/3, SOX2,SSEA-1, SSEA-3, SSEA-4, STAT3, Stella/Dppa3, SUZ12, TBX2, TBX3, TBX5,TERT, TEX19, TEX19.1, THAP11, TRA-1-60(R), TROP-2, UTF1, and/or ZIC3. Ina specific example, the expressed pluripotency marker is alkalinephosphatase, NANOG, or both.

In another embodiment, human iPS cells cultured in the low osmolalitymedium described herein display morphological characteristics indicativeof a naïve state. An exemplary morphology is characterized by cellshaving compact dome-shaped colonies in culture.

The human iPS cells cultured in the low osmolality medium describedherein can have a normal karyotype. A normal karyotype includes akaryotype in which all chromosomes normally characteristic of thespecies are present and have not been noticeably altered or a state ofcells lacking any visible numerical or structural chromosomalabnormality detectable with chromosome banding analysis. The human iPScells cultured in the low osmolality medium described herein can have anormal karyotype, for example, after about 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 passages in thelow osmolality medium described herein.

In another embodiment, human iPS cells cultured in the low osmolalitymedium described herein can be mechanically or enzymatically dissociatedinto a single-cell suspension, passaged, and/or subcultured. Such humaniPS cells cultured in the low osmolality medium described herein canhave a normal karyotype and can maintain the normal karyotype afterbeing mechanically or enzymatically dissociated into a single-cellsuspension, passaged, and/or subcultured. For example, such human iPScells cultured in the low osmolality medium described herein can have anormal karyotype and can maintain the normal karyotype after beingmechanically or enzymatically dissociated into a single-cell suspension,modified at a target genomic locus using the methods described elsewhereherein, and subcultured. In one example, enzymatic dissociation can beperformed using trypsin.

When cultured in the present low osmolality medium, human iPS cells canprovide greater transformation efficiency due to enhanced dissociationinto a single-cell suspension. With other types of medium (e.g., mTeSR™medium or 2i medium) typically used to maintain human iPS cells inculture, dissociation of human iPS cells must be performed mechanicallyor with enzymes such as collagenase that are less harsh than trypsin. Itis generally not recommended to passage human iPS cells as single cells,as this practice has been demonstrated to place unwanted selectivepressures on cell populations that can lead to, for example, geneticaberrations in culture. Human iPS cells are vulnerable to apoptosis uponcellular detachment and dissociation, and typically undergo massive celldeath after complete dissociation. See Watanabe et al. (2007) Nature25(6):681-686, herein incorporated by reference in its entirety for allpurposes. Thus, dissociation of human iPS cells is typically performedwith reagents or methods that minimize the breakup of colonies whenpassaging and do not create single-cell suspensions. Consequently, thecells are not dissociated as effectively or as completely. However,complete dissociation can be important for procedures such as clonalisolation following gene transfer or generation of a targeted geneticmodification, particularly when attempting to isolate relatively rareclones such as those undergoing homologous recombination to produce adesired targeted modification. In contrast, with the present lowosmolality medium, trypsin can be used to dissociate the cells, and theenhanced dissociation results in increased transformation efficiency.For example, such dissociation can create single-cell suspensions thatresult in greater targeting efficiencies when targeting, for example,via electroporation or using the methods for making targeted geneticmodifications described elsewhere herein. Furthermore, unlike with othertypes of medium typically used to maintain human iPS cells in culture(e.g., mTeSR™ medium or 2i medium), enzymatic dissociation of human iPScells cultured with the present low osmolality medium (preferably a lowosmolality medium not comprising bFGF) can be performed in the absenceof one or more inhibitors that are generally necessary for the passageof such cells. An exemplary inhibitor that can be omitted is aRho-associated protein kinase (ROCK) inhibitor. A ROCK inhibitor isgenerally necessary when passaging human iPS cells to inhibit theactivation of pro-apoptotic pathways. In particular, addition of a ROCKinhibitor is generally recommended when plating single-cell suspensionsof human iPS cells, as this has been reported to increase cell survival.See Watanabe et al. (2007) Nature 25(6):681-686. When using the lowosmolality medium disclosed herein, however, such ROCK inhibitors arenot needed, even when passaging as single-cell suspensions. Suchsingle-cell suspensions can maintain pluripotency and a normal karyotypefollowing trypsinization and replating when the low osmolality mediumdisclosed herein is used.

In a further embodiment, subcultured human iPS cells cultured in the lowosmolality medium described herein can maintain a naïve or naïve-lookingstate following enzymatic dissociation and subculture. Subcultured humaniPS cells cultured in the low osmolality medium described herein canmaintain a naïve or naïve-looking state following enzymatic dissociationand subculture even when passaged as single-cell suspensions and/or whenmodified at a target genomic locus using the methods described elsewhereherein. In some examples, subcultured human iPS cells can continue todisplay a morphology characterized by compact dome-shaped colonies.Subcultured human iPS cells can also continue to express one orpluripotency markers as described herein.

C. Methods of Making and Maintaining a Population of Human InducedPluripotent Stem Cells

Methods and compositions are provided for making human iPS cells in anin vitro culture. Methods and compositions are further provided formaintaining human iPS cells in an in vitro culture.

The term “making” includes culturing non-pluripotent cells transformedto express one or more reprogramming factors as described herein, undersuitable conditions to induce a change in cell phenotype, geneexpression, or both, such that the cells display a naïve ornaïve-looking state, i.e., express one or more characteristics of naïvehuman iPS cells. A naïve or naïve-looking state can be expressed inresponse to particular culture conditions, e.g., culture in a lowosmolality medium as described herein. In some examples, the proportionof cells expressing a naïve or naïve-looking state is at least about30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and up to 100% of the cells inculture.

In one embodiment, the method enriches an in vitro culture for apopulation of naïve or naïve-looking human iPS cells. In such anembodiment, naïve or naïve-looking human iPS cells can be propagated inculture preferentially over cells that do not express a naïve ornaïve-looking state. In another embodiment, naïve or naïve-looking humaniPS cells can be selected from a culture, be enzymatically dissociated,and subcultured to produce an enriched population of naïve ornaïve-looking human iPS cells.

In one embodiment, non-pluripotent cells transformed to express apluripotent state, are cultured in vitro in a medium provided hereinthat is suitable for inducing expression of a naïve or naïve-lookingstate for a period of at least 1, 2, 5, 7, 10, 14, 21, or 28 days, orany period of time sufficient to induce expression of a naïve ornaïve-looking state in culture. Transformed cells can be cultured in thepresent medium for at least 1, 2, 3, or 4 weeks. Sometimes transformedcells are cultured for 1-4 weeks. Expression of a naïve or naïve-lookingstate can be determined by observing morphological characteristics orthe expression of pluripotency markers, characteristic of a naïve ornaïve-looking state, that are described elsewhere herein.

In one embodiment, non-pluripotent cells transformed to express apluripotent state, are cultured in the present low osmolality mediumuntil they express characteristics of a naïve or naïve-looking state.Cells can then be cultured in the present medium to maintain a naïve ornaïve-looking state. In another embodiment, non-pluripotent cellstransformed to express a pluripotent state, are first cultured in a highosmolality medium prior to culturing in the present low osmolalitymedium. Such high osmolality medium exhibits an osmolality higher thanthe present low osmolality medium and can comprise bFGF. The osmolalityof the high osmolality medium can be, for example, about 300-380,310-370, 320-360, 330-350, or 340 mOsm/kg. Alternatively, the osmolalityof the high osmolality medium can be, for example, 340±70, 340±60,340±50, 340±40, 340±30, 340±20, or 340±10 mOsm/kg. For example, theosmolality of the high osmolality medium can be about 270-280, 280-290,290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370,370-380, 380-390, 390-400, or 400-410 mOsm/kg. Alternatively, theosmolality of the high osmolality medium can be at least about 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or 410mOsm/kg. Some high osmolality medium comprises one or more of bovineserum albumin, bFGF, transforming growth factor β (TGFβ), lithiumchloride, pipecolic acid, and gamma-aminobutyric acid (GABA). Examplesof a high osmolality medium include mTeSR™ medium (StemcellTechnologies).

In some embodiments, non-pluripotent cells transformed to express apluripotent state, can first be cultured in high osmolality mediumcomprising bFGF until they begin to express characteristics of a naïveor naïve-looking state, at which time the cells are cultured in thepresent low osmolality medium. In one example, cells can be cultured inhigh osmolality medium comprising bFGF for a period of at least 1, 2, 5,10, 30, 60, or 90 days, a period of 1, 2, 4, 8, or 12 weeks, or a periodbetween 1 day to 3 months. An exemplary time period for culture in ahigh osmolality medium comprising bFGF is 2 months.

In other embodiments, non-pluripotent cells transformed to express apluripotent state, can first be cultured in high osmolality mediumcomprising bFGF until they begin to display a morphology characterizedby three-dimensional cell clumps, at which time cells are cultured inthe present low osmolality medium. In such embodiments, cells displayingthree-dimensional clumps can be selected, dissociated (e.g., withtrypsin), and transferred to a new culture in the low osmolality mediumdescribed herein.

The terms “maintain,” “maintaining,” and “maintenance” include thepreservation of at least one or more of the characteristics orphenotypes of the human iPS cells described herein. Such characteristicscan include maintaining pluripotency, cell morphology, gene expressionprofiles, and/or other functional characteristics of naïve cells. Theterms “maintain,” “maintaining,” and “maintenance” can also encompassthe propagation of cells and/or an increase in the number of naïve cellsbeing cultured. The terms include culture conditions that prevent cellsfrom converting to a primed or non-pluripotent state. The terms furtherinclude culture conditions that permit the cells to remain pluripotentand/or naïve, while the cells may or may not continue to divide andincrease in number.

In one embodiment, human iPS cells are cultured in vitro in a mediumprovided herein that is suitable for maintaining such cells in a naïveor naïve-looking state. In a particular example, human iPS cells can becultured in a suitable medium for a period of 1, 2, 5, 7, 10, 14, 21, or28 days, or for a period of about 2 weeks, about 3 weeks, about 4 weeks,or more, so long as the cultured cells are maintained in a naïve ornaïve-looking state. Cells can be cultured for at least 1, 2, 3 or 4weeks. Sometimes cells are cultured for 1-4 weeks. Human iPS cells canbe maintained, for example, for any period of time sufficient forpropagation of the cells in culture, genetic modification of the cells,and/or subculture of the cells.

In another embodiment, human iPS cells or non-pluripotent cellstransformed to express a pluripotent state, can be cultured on asubstrate or feeder cell layer suitable for in vitro culture. In aparticular example, cells are cultured on MATRIGEL™ (BD Biosciences). Inanother example, cells are cultured on newborn human foreskin fibroblast(NuFF) feeder cells. In another example, cells are cultured on GELTREX™(Life Technologies). In another example, the cells are cultured onvitronectin (e.g., VITRONECTIN XF™ (STEMCELL Technologies).

In a further embodiment, the doubling time of human iPS cells culturedin the present low osmolality medium is reduced as compared to primedhuman iPS cells or non-pluripotent cells transformed to express apluripotent state. In a particular example, the doubling time of thepresent human iPS cells is between about 16-24 hours.

D. Genetic Modifications and Methods for Making Targeted GeneticModifications

In some embodiments, the methods for making and maintaining human iPScells comprise introducing a genetic modification into the human iPScells. Likewise, the invention provides human iPS cells that comprise agenetic modification.

In particular embodiments, the genetic modification comprises amodification of one or more endogenous nucleic acids, a substitution ofone or more endogenous nucleic acids, a replacement of an endogenousnucleic acid with a heterologous nucleic acid, a knockout, or aknock-in. In specific examples, the genetic modification is introducedby introducing a large targeting vector (LTVEC) into the human iPS cellsor the non-pluripotent cells transformed to express a pluripotent state.In such an example, the LTVEC can comprise DNA to be inserted into thegenome of the cells.

Various methods for making targeted genetic modifications in human iPScells can be used. For example, various methods for making targetedgenetic modifications that modify the level and/or the activity ofproteins in human iPS cells can be used. For example, in one instance,the targeted genetic modification employs a system that will generate atargeted genetic modification via a homologous recombination (HR) event.Homology-directed repair (HDR) or HR includes a form of nucleic acidrepair that can require nucleotide sequence homology, uses a “donor”molecule to template repair of a “target” molecule (i.e., the one thatexperienced the double-strand break), and leads to transfer of geneticinformation from the donor to target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or synthesis-dependent strand annealing, in which the donor is usedto resynthesize genetic information that will become part of the target,and/or related processes. In some cases, the donor polynucleotide, aportion of the donor polynucleotide, a copy of the donor polynucleotide,or a portion of a copy of the donor polynucleotide integrates into thetarget DNA. In other instances, a cell can be modified using nucleaseagents that generate a single or double strand break at a targetedgenomic location. The single or double-strand break is then repaired bythe non-homologous end joining pathway (NHEJ). NHEJ includes the repairof double-strand breaks in a nucleic acid by direct ligation of thebreak ends to one another without the need for a homologous template.Ligation of non-contiguous sequences by NHEJ can often result indeletions, insertions, or translocations near the site of thedouble-strand break. Such systems find use, for example, in generatingtargeted loss of function genetic modifications. Non-limiting methodsfor generating such targeted genetic modification are discussed indetail elsewhere herein, including, for example, the use of targetingplasmids or large targeting vectors. See, also, Wang et al. (2013) Cell153:910-918, Mandalos et al. (2012) PLOS ONE 7:e45768:1-9, and Wang etal. (2013) Nat Biotechnol. 31:530-532, each of which is hereinincorporated by reference.

It is recognized that in specific embodiments, the targeted geneticmodification of any polynucleotide of interest can occur while thepluripotent cell (i.e., human iPS cell) is being maintained in theculture medium described herein. Alternatively, the targeted geneticmodification of any polynucleotide of interest can occur while thepluripotent cell (i.e., human iPS cell) is being maintained in differentculture medium, and subsequently transferred to the low osmolalityculture medium disclosed herein.

In general, the level and/or activity of a protein is modified if theprotein level and/or the activity level of the protein is statisticallyhigher or lower than the protein level in an appropriate control cellthat has not been genetically modified or mutagenized to alter theexpression and/or activity of the protein. In specific embodiments, theconcentration and/or activity of the protein is altered by at least 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a controlcell which has not been modified to have a modified level and/oractivity of the protein.

A “subject cell” is one in which a genetic alteration, such as a geneticmodification disclosed herein has been effected, or is a cell which isdescended from a cell so altered and which comprises the alteration. A“control” or “control cell” provides a reference point for measuringchanges in phenotype of the subject cell. In one embodiment, a controlcell is as closely matched as possible with the cell with reducedprotein activity except it lacks the genetic modification or mutationresulting in the modified activity (for example, the respective cellscan originate from the same cell line). In other instances, the controlcell may comprise, for example: (a) a wild-type cell, i.e., of the samegenotype as the starting material for the genetic alteration whichresulted in the subject cell; (b) a cell of the same genotype as thestarting material but which has been genetically modified with a nullconstruct (i.e. with a construct which has no known effect on the traitof interest, such as a construct comprising a marker gene); (c) a cellwhich is a non-genetically modified progeny of a subject cell (i.e., thecontrol cell and the subject cell originate from the same cell line);(d) a cell genetically identical to the subject cell but which is notexposed to conditions or stimuli that would induce expression of thegene of interest; or (e) the subject cell itself, under conditions inwhich the genetic modification does not result in an alteration inexpression of the polynucleotide of interest.

The expression level of the polypeptide may be measured directly, forexample, by assaying for the level of the polypeptide in the cell ororganism, or indirectly, for example, by measuring the activity of thepolypeptide.

In other instances, cells having the targeted genetic modification areselected using methods that include, but are not limited to, Southernblot analysis, DNA sequencing, PCR analysis, or phenotypic analysis.Such cells are then employed in the various methods and compositionsdescribed herein.

A targeted genetic modification can comprise a targeted alteration to apolynucleotide of interest. Such targeted modifications include, but arenot limited to, additions of one or more nucleotides, deletions of oneor more nucleotides, substitutions of one or more nucleotides, aknockout of the polynucleotide of interest or a portion thereof, aknock-in of the polynucleotide of interest or a portion thereof, areplacement of an endogenous nucleic acid sequence with a heterologousnucleic acid sequence, or a combination thereof. In specificembodiments, at least 1, 2, 3, 4, 5, 7, 8, 9, 10, 100, 500 or morenucleotides or at least 10 kb to 500 kb or more are changed to form thetargeted genomic modification.

In other embodiments, the activity and/or level of a polypeptide ismodified by introducing into the cell a polynucleotide that alters thelevel or activity of the polypeptide. The polynucleotide may modify theexpression of the polypeptide directly, by altering translation of themessenger RNA, or indirectly, by encoding a polypeptide that alters thetranscription or translation of the gene encoding a protein. In otherembodiments, the activity of a polypeptide is modified by introducinginto the cell a sequence encoding a polypeptide that alters the activityof the target polypeptide.

In one embodiment, human iPS cells can comprise a conditional allelethat modifies the activity and/or level of a protein. A “conditionalallele” includes a modified gene designed to have the modified leveland/or activity of the protein at a desired developmental time and/orwithin a desired tissue of interest. The modified level and/or activitycan be compared with a control cell lacking the modification giving riseto the conditional allele, or in the case of modified activity at adesired developmental time with preceding and/or following times, or inthe case of a desired tissue, with a mean activity of all tissues. Inone embodiment, the conditional allele comprises a conditional nullallele of the gene that can be switched off or on at a desireddevelopmental time point and/or in specific tissues.

In a non-limiting embodiment, the conditional allele is amultifunctional allele, as described in US 2011/0104799, which isincorporated by reference in its entirety. In specific embodiments, theconditional allele comprises: (a) an actuating sequence in senseorientation with respect to transcription of a target gene, and a drugselection cassette (DSC) in sense or antisense orientation; (b) inantisense orientation a nucleotide sequence of interest (NSI) and aconditional by inversion module (COIN, which utilizes an exon-splittingintron and an invertible genetrap-like module; see, for example, US2011/0104799, which is incorporated by reference in its entirety); and(c) recombinable units that recombine upon exposure to a firstrecombinase to form a conditional allele that (i) lacks the actuatingsequence and the DSC, and (ii) contains the NSI in sense orientation andthe COIN in antisense orientation.

The present invention allows for modifying a target genomic locus on achromosome in a cell. In particular embodiments, the methods providedherein allow for the targeting of a genomic locus on a chromosome byemploying a targeting vector in the absence of, or in combination with,a nuclease agent.

Methods for making targeted genetic modifications can comprise, forexample, the use of a targeting vector (e.g., an LTVEC), either alone orin combination with one or more nucleases as described elsewhere herein.See, e.g., US 2015/0159175, US 2015/0159174, US 2014/0310828, US2014/0309487, and US 2013/0309670, each of which is herein incorporatedby reference in its entirety for all purposes. Likewise, methods formaking targeted genetic modifications can comprise the use of one ormore nucleases either alone or in combination with a targeting vector.

For example, methods are provided for modifying a target genomic locusin a human iPS cell, comprising: (a) introducing into the cell one ormore nuclease agents that induces one or more nicks or double-strandbreaks at a recognition site at or near the target genomic locus; and(b) identifying at least one cell comprising in its genome amodification at the target genomic locus. Such methods can result indisruption of the target genomic locus. Disruption of the endogenousnucleic acid sequence can result, for example, when a double-strandbreak created by a nuclease is repaired by non-homologous end joining(NHEJ)-mediated DNA repair, which generates a mutant allele comprisingan insertion or a deletion of a nucleic acid sequence and thereby causesdisruption of that genomic locus. Examples of disruption includealteration of a regulatory element (e.g., promoter or enhancer), amissense mutation, a nonsense mutation, a frame-shift mutation, atruncation mutation, a null mutation, or an insertion or deletion ofsmall number of nucleotides (e.g., causing a frameshift mutation).Disruption can result in inactivation (i.e., loss of function) or lossof the allele.

Other methods for modifying a target genomic locus in a human iPS cellcomprise: (a) introducing into the cell a targeting vector comprising aninsert nucleic acid flanked by 5′ and 3′ homology arms corresponding to5′ and 3′ target sites; and (b) identifying at least one cell comprisingin its genome the insert nucleic acid integrated at the target genomiclocus.

Other methods for modifying a target genomic locus in a human iPS cellcomprise: (a) introducing into the cell: (i) a nuclease agent, whereinthe nuclease agent induces a nick or double-strand break at arecognition site within the target genomic locus; and (ii) a targetingvector comprising an insert nucleic acid flanked by 5′ and 3′ homologyarms corresponding to 5′ and 3′ target sites located in sufficientproximity to the recognition site; and (c) identifying at least one cellcomprising a modification (e.g., integration of the insert nucleic acid)at the target genomic locus. Such methods can result in various types oftargeted genetic modifications. Such targeted modifications can include,for example, additions of one or more nucleotides, deletions of one ormore nucleotides, substitutions of one or more nucleotides, a pointmutation, a knockout of a polynucleotide of interest or a portionthereof, a knock-in of a polynucleotide of interest or a portionthereof, a replacement of an endogenous nucleic acid sequence with aheterologous, exogenous, or orthologous nucleic acid sequence, a domainswap, an exon swap, an intron swap, a regulatory sequence swap, a geneswap, or a combination thereof. For example, at least 1, 2, 3, 4, 5, 7,8, 9, 10 or more nucleotides can be changed to form the targeted genomicmodification. The deletions, insertions, or replacements can be of anysize, as disclosed elsewhere herein.

a. Nuclease Agents and Recognition Sites for Nuclease Agents

The term “recognition site for a nuclease agent” includes a DNA sequenceat which a nick or double-strand break is induced by a nuclease agent.The recognition site for a nuclease agent can be endogenous (or native)to the cell or the recognition site can be exogenous to the cell. Inspecific embodiments, the recognition site is exogenous to the cell andthereby is not naturally occurring in the genome of the cell. In stillfurther embodiments, the recognition site is exogenous to the cell andto the polynucleotides of interest that one desired to be positioned atthe target locus. In further embodiments, the exogenous or endogenousrecognition site is present only once in the genome of the host cell. Inspecific embodiments, an endogenous or native site that occurs only oncewithin the genome is identified. Such a site can then be used to designnuclease agents that will produce a nick or double-strand break at theendogenous recognition site.

The length of the recognition site can vary and includes, for example,recognition sites that are about 30-36 by for a zinc finger nuclease(ZFN) pair (i.e., about 15-18 by for each ZFN), about 36 by for aTranscription Activator-Like Effector Nuclease (TALEN), or about 20 byfor a CRISPR/Cas9 guide RNA.

In one embodiment, each monomer of the nuclease agent recognizes arecognition site of at least 9 nucleotides. In other embodiments, therecognition site is from about 9 to about 12 nucleotides in length, fromabout 12 to about 15 nucleotides in length, from about 15 to about 18nucleotides in length, or from about 18 to about 21 nucleotides inlength, and any combination of such subranges (e.g., 9-18 nucleotides).It is recognized that a given nuclease agent can bind the recognitionsite and cleave that binding site or alternatively, the nuclease agentcan bind to a sequence that is different from the recognition site.Moreover, the term recognition site comprises both the nuclease agentbinding site and the nick/cleavage site irrespective whether thenick/cleavage site is within or outside the nuclease agent binding site.In another variation, the cleavage by the nuclease agent can occur atnucleotide positions immediately opposite each other to produce a bluntend cut or, in other cases, the incisions can be staggered to producesingle-stranded overhangs, also called “sticky ends”, which can beeither 5′ overhangs, or 3′ overhangs.

Any nuclease agent that induces a nick or double-strand break into adesired recognition site can be used in the methods and compositionsdisclosed herein. A naturally-occurring or native nuclease agent can beemployed so long as the nuclease agent induces a nick or double-strandbreak in a desired recognition site. Alternatively, a modified orengineered nuclease agent can be employed. An “engineered nucleaseagent” includes a nuclease that is engineered (modified or derived) fromits native form to specifically recognize and induce a nick ordouble-strand break in the desired recognition site. Thus, an engineerednuclease agent can be derived from a native, naturally-occurringnuclease agent or it can be artificially created or synthesized. Themodification of the nuclease agent can be as little as one amino acid ina protein cleavage agent or one nucleotide in a nucleic acid cleavageagent. In some embodiments, the engineered nuclease induces a nick ordouble-strand break in a recognition site, wherein the recognition sitewas not a sequence that would have been recognized by a native(non-engineered or non-modified) nuclease agent. Producing a nick ordouble-strand break in a recognition site or other DNA can be referredto herein as “cutting” or “cleaving” the recognition site or other DNA.

Active variants and fragments of the exemplified recognition sites arealso provided. Such active variants can comprise at least 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the given recognition site, wherein the activevariants retain biological activity and hence are capable of beingrecognized and cleaved by a nuclease agent in a sequence-specific mannerAssays to measure the double-strand break of a recognition site by anuclease agent are known in the art (e.g., TaqMan® qPCR assay, FrendeweyD. et al., Methods in Enzymology, 2010, 476:295-307, which isincorporated by reference herein in its entirety).

The recognition site of the nuclease agent can be positioned anywhere inor near the target locus. The recognition site can be located within acoding region of a gene, or within regulatory regions that influenceexpression of the gene. A recognition site of the nuclease agent can belocated in an intron, an exon, a promoter, an enhancer, a regulatoryregion, or any non-protein coding region. In specific embodiments, therecognition site is positioned within the polynucleotide encoding theselection marker. Such a position can be located within the codingregion of the selection marker or within the regulatory regions, whichinfluence the expression of the selection marker. Thus, a recognitionsite of the nuclease agent can be located in an intron of the selectionmarker, a promoter, an enhancer, a regulatory region, or anynon-protein-coding region of the polynucleotide encoding the selectionmarker. In specific embodiments, a nick or double-strand break at therecognition site disrupts the activity of the selection marker. Methodsto assay for the presence or absence of a functional selection markerare known.

In one embodiment, the nuclease agent is a Transcription Activator-LikeEffector Nuclease (TALEN). TAL effector nucleases are a class ofsequence-specific nucleases that can be used to make double-strandbreaks at specific target sequences in the genome of a prokaryotic oreukaryotic organism. TAL effector nucleases are created by fusing anative or engineered transcription activator-like (TAL) effector, orfunctional part thereof, to the catalytic domain of an endonuclease,such as, for example, FokI. The unique, modular TAL effector DNA bindingdomain allows for the design of proteins with potentially any given DNArecognition specificity. Thus, the DNA binding domains of the TALeffector nucleases can be engineered to recognize specific DNA targetsites and thus, used to make double-strand breaks at desired targetsequences. See, WO 2010/079430; Morbitzer et al. (2010) PNAS10.1073/pnas.1013133107; Scholze & Boch (2010) Virulence 1:428-432;Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc.Acids Res. (2010) doi:10.1093/nar/gkq704; and Miller et al. (2011)Nature Biotechnology 29:143-148; all of which are herein incorporated byreference.

Examples of suitable TAL nucleases, and methods for preparing suitableTAL nucleases, are disclosed, e.g., in US Patent Application No.2011/0239315 A1, 2011/0269234 A1, 2011/0145940 A1, 2003/0232410 A1,2005/0208489 A1, 2005/0026157 A1, 2005/0064474 A1, 2006/0188987 A1, and2006/0063231 A1 (each hereby incorporated by reference). In variousembodiments, TAL effector nucleases are engineered that cut in or near atarget nucleic acid sequence in, e.g., a locus of interest or a genomiclocus of interest, wherein the target nucleic acid sequence is at ornear a sequence to be modified by a targeting vector. The TAL nucleasessuitable for use with the various methods and compositions providedherein include those that are specifically designed to bind at or neartarget nucleic acid sequences to be modified by targeting vectors asdescribed herein.

In one embodiment, each monomer of the TALEN comprises 12-25 TALrepeats, wherein each TAL repeat binds a 1 by subsite. In some TALENs,each monomer of the TALEN comprises 33-35 TAL repeats that recognize asingle base pair via two hypervariable residues. In one embodiment, thenuclease agent is a chimeric protein comprising a TAL repeat-based DNAbinding domain operably linked to an independent nuclease. In oneembodiment, the independent nuclease is a FokI endonuclease. In oneembodiment, the nuclease agent comprises a first TAL-repeat-based DNAbinding domain and a second TAL-repeat-based DNA binding domain, whereineach of the first and the second TAL-repeat-based DNA binding domains isoperably linked to a FokI nuclease, wherein the first and the secondTAL-repeat-based DNA binding domain recognize two contiguous target DNAsequences in each strand of the target DNA sequence separated by about 6by to about 40 by cleavage site, and wherein the Fold nucleases dimerizeand make a double strand break at a target sequence. For example, thenuclease agent can comprise a first TAL-repeat-based DNA binding domainand a second TAL-repeat-based DNA binding domain, wherein each of thefirst and the second TAL-repeat-based DNA binding domains is operablylinked to a FokI nuclease, wherein the first and the secondTAL-repeat-based DNA binding domain recognize two contiguous target DNAsequences in each strand of the target DNA sequence separated by aspacer sequence of varying length (12-20 bp), and wherein the Foldnuclease subunits dimerize to create an active nuclease that makes adouble strand break at a target sequence.

The nuclease agent employed in the various methods and compositionsdisclosed herein can further comprise a zinc-finger nuclease (ZFN). Inone embodiment, each monomer of the ZFN comprises 3 or more zincfinger-based DNA binding domains, wherein each zinc finger-based DNAbinding domain binds to a 3 by subsite. In other embodiments, the ZFN isa chimeric protein comprising a zinc finger-based DNA binding domainoperably linked to an independent nuclease. In one embodiment, theindependent endonuclease is a FokI endonuclease. In one embodiment, thenuclease agent comprises a first ZFN and a second ZFN, wherein each ofthe first ZFN and the second ZFN is operably linked to a Fold nuclease,wherein the first and the second ZFN recognize two contiguous target DNAsequences in each strand of the target DNA sequence separated by about 6by to about 40 by cleavage site, and wherein the Fold nucleases dimerizeand make a double strand break. For example, the nuclease agent cancomprise a first ZFN and a second ZFN, wherein each of the first ZFN andthe second ZFN is operably linked to a FokI nuclease subunit, whereinthe first and the second ZFN recognize two contiguous target DNAsequences in each strand of the target DNA sequence separated by about5-7 by spacer, and wherein the Fold nuclease subunits dimerize to createan active nuclease that makes a double strand break. See, for example,US20060246567; US20080182332; US20020081614; US20030021776;WO/2002/057308A2; US20130123484; US20100291048; and, WO/2011/017293A2,each of which is herein incorporated by reference.

In still another embodiment, the nuclease agent is a meganuclease.Meganucleases have been classified into four families based on conservedsequence motifs, the families are the LAGLIDADG, GIY-YIG, H-N-H, andHis-Cys box families. These motifs participate in the coordination ofmetal ions and hydrolysis of phosphodiester bonds. Meganucleases arenotable for their long recognition sites, and for tolerating somesequence polymorphisms in their DNA substrates. Meganuclease domains,structure and function are known, see for example, Guhan and Muniyappa(2003) Crit Rev Biochem Mol Biol 38:199-248; Lucas et al., (2001)Nucleic Acids Res 29:960-9; Jurica and Stoddard, (1999) Cell Mol LifeSci 55:1304-26; Stoddard, (2006) Q Rev Biophys 38:49-95; and Moure etal., (2002) Nat Struct Biol 9:764. In some examples a naturallyoccurring variant, and/or engineered derivative meganuclease is used.Methods for modifying the kinetics, cofactor interactions, expression,optimal conditions, and/or recognition site specificity, and screeningfor activity are known, see for example, Epinat et al., (2003) NucleicAcids Res 31:2952-62; Chevalier et al., (2002) Mol Cell 10:895-905;Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et al., (2002)Nucleic Acids Res 30:3870-9; Sussman et al., (2004) J Mol Biol342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; Chames etal., (2005) Nucleic Acids Res 33:e178; Smith et al., (2006) NucleicAcids Res 34:e149; Gruen et al., (2002) Nucleic Acids Res 30:e29; Chenand Zhao, (2005) Nucleic Acids Res 33:e154; WO2005105989; WO2003078619;WO2006097854; WO2006097853; WO2006097784; and WO2004031346.

Any meganuclease can be used herein, including, but not limited to,I-Seel, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI,I-CeuAIIP, I-CreI, I-CrepsblP, I-CrepsbllP, I-CrepsbIIIP, I-CrepsblVP,I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII,I-Amal, I-Anil, I-ChuI, I-Cmoel, I-CpaI, I-CpaII, I-CsmI, I-CvuI,I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP,I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-NjaI,I-Nsp236IP, I-Paid, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP,I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-Seal, I-SexIP,I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I,I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII,I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI,PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII,PI-Rma43812IP, PI-SpBetaIP, PI-Seel, PI-TfuI, PI-TfuII, PI-Thyl,PI-TliI, PI-TliII, or any active variants or fragments thereof.

In one embodiment, the meganuclease recognizes double-stranded DNAsequences of 12 to 40 base pairs. In one embodiment, the meganucleaserecognizes one perfectly matched target sequence in the genome. In oneembodiment, the meganuclease is a homing nuclease. In one embodiment,the homing nuclease is a LAGLIDADG family of homing nuclease. In oneembodiment, the LAGLIDADG family of homing nuclease is selected fromI-SeeI, I-CreI, and I-Dmol.

Nuclease agents can further comprise restriction endonucleases, whichinclude Type I, Type II, Type III, and Type IV endonucleases. Type I andType III restriction endonucleases recognize specific recognition sites,but typically cleave at a variable position from the nuclease bindingsite, which can be hundreds of base pairs away from the cleavage site(recognition site). In Type II systems the restriction activity isindependent of any methylase activity, and cleavage typically occurs atspecific sites within or near to the binding site. Most Type II enzymescut palindromic sequences, however Type IIa enzymes recognizenon-palindromic recognition sites and cleave outside of the recognitionsite, Type IIb enzymes cut sequences twice with both sites outside ofthe recognition site, and Type IIs enzymes recognize an asymmetricrecognition site and cleave on one side and at a defined distance ofabout 1-20 nucleotides from the recognition site. Type IV restrictionenzymes target methylated DNA. Restriction enzymes are further describedand classified, for example in the REBASE database (webpage atrebase.neb.com; Roberts et al., (2003) Nucleic Acids Res 31:418-20),Roberts et al., (2003) Nucleic Acids Res 31:1805-12, and Belfort et al.,(2002) in Mobile DNA II, pp. 761-783, Eds. Craigie et al., (ASM Press,Washington, D.C.).

The nuclease agent employed in the various methods and compositions canalso comprise a Clustered Regularly Interspersed Short PalindromicRepeats (CRISPR)/CRISPR-associated (Cas) system or components of such asystem. CRISPR/Cas systems include transcripts and other elementsinvolved in the expression of, or directing the activity of, Cas genes.A CRISPR/Cas system can be a type I, a type II, or a type III system.The methods and compositions disclosed herein employ CRISPR/Cas systemsby utilizing CRISPR complexes (comprising a guide RNA (gRNA) complexedwith a Cas protein) for site-directed cleavage of nucleic acids.

Some CRISPR/Cas systems used in the methods disclosed herein arenon-naturally occurring. A “non-naturally occurring” system includesanything indicating the involvement of the hand of man, such as one ormore components of the system being altered or mutated from theirnaturally occurring state, being at least substantially free from atleast one other component with which they are naturally associated innature, or being associated with at least one other component with whichthey are not naturally associated. For example, some CRISPR/Cas systemsemploy non-naturally occurring CRISPR complexes comprising a gRNA and aCas protein that do not naturally occur together.

Cas proteins generally comprise at least one RNA recognition or bindingdomain. Such domains can interact with guide RNAs (gRNAs, described inmore detail below). Cas proteins can also comprise nuclease domains(e.g., DNase or RNase domains), DNA binding domains, helicase domains,protein-protein interaction domains, dimerization domains, and otherdomains. A nuclease domain possesses catalytic activity for nucleic acidcleavage. Cleavage includes the breakage of the covalent bonds of anucleic acid molecule. Cleavage can produce blunt ends or staggeredends, and it can be single-stranded or double-stranded.

Examples of Cas proteins include Casl, Cas1B, Cas2, Cas3, Cas4, CasS,Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c,Cas9 (Csnl or Csx12), Cas10, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3,Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, CsaS,Csn2, Csm2, Csm3, Csm4, CsmS, Csm6, Cmrl , Cmr3, Cmr4, CmrS, Cmr6, Csb1,Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx15, Csf1,Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof.

In some instances, a Cas protein is from a type II CRISPR/Cas system.For example, the Cas protein can be a Cas9 protein or be derived from aCas9 protein. Cas9 proteins typically share four key motifs with aconserved architecture. Motifs 1, 2, and 4 are RuvC-like motifs, andmotif 3 is an HNH motif. The Cas9 protein can be from, for example,Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp.,Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomycesviridochromogenes, Streptomyces viridochromogenes, Streptosporangiumroseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius,Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacteriumsibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius,Microscilla marina, Burkholderiales bacterium, Polaromonasnaphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothecesp., Microcystis aeruginosa, Synechococcus sp., Acetohalobiumarabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, CandidatusDesulforudis, Clostridium botulinum, Clostridium difficile, Finegoldiamagna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum,Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatiumvinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcuswatsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer,Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena,Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.,Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotogamobilis, Thermosipho africanus, or Acaryochloris marina. The Cas9protein can be from Staphylococcus aureus. Additional examples of theCas9 family members include those described in WO 2014/131833, hereinincorporated by reference in its entirety. In a specific example, theCas9 protein is a Cas9 protein from S. pyogenes or is derived therefrom.The amino acid sequence of a Cas9 protein from S. pyogenes can be found,for example, in the SwissProt database under accession number Q99ZW2.

Cas proteins can be wild type proteins (i.e., those that occur innature), modified Cas proteins (i.e., Cas protein variants), orfragments of wild type or modified Cas proteins. Cas proteins can alsobe active variants or fragments of wild type or modified Cas proteins.Active variants or fragments can comprise at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thewild type or modified Cas protein or a portion thereof, wherein theactive variants retain the ability to cut at a desired cleavage site andhence retain nick-inducing or double-strand-break-inducing activity.Assays for nick-inducing or double-strand-break-inducing activity areknown and generally measure the overall activity and specificity of theCas protein on DNA substrates containing the cleavage site.

Cas proteins can be modified to increase or decrease nucleic acidbinding affinity, nucleic acid binding specificity, and/or enzymaticactivity. Cas proteins can also be modified to change any other activityor property of the protein, such as stability. For example, one or morenuclease domains of the Cas protein can be modified, deleted, orinactivated, or a Cas protein can be truncated to remove domains thatare not essential for the function of the protein or to optimize (e.g.,enhance or reduce) the activity of the Cas protein.

Some Cas proteins comprise at least two nuclease domains, such as DNasedomains. For example, a Cas9 protein can comprise a RuvC-like nucleasedomain and an HNH-like nuclease domain. The RuvC and HNH domains caneach cut a different strand of double-stranded DNA to make adouble-stranded break in the DNA. See, e.g., Jinek et al. (2012) Science337:816-821, hereby incorporated by reference in its entirety.

One or both of the nuclease domains can be deleted or mutated so thatthey are no longer functional or have reduced nuclease activity. If oneof the nuclease domains is deleted or mutated, the resulting Cas protein(e.g., Cas9) can be referred to as a nickase and can generate a singlestrand break at a target sequence within a double-stranded DNA but not adouble strand break (i.e., it can cleave the complementary strand or thenon-complementary strand, but not both). If both of the nuclease domainsare deleted or mutated, the resulting Cas protein (e.g., Cas9) will havea reduced ability to cleave both strands of a double-stranded DNA. Anexample of a mutation that converts Cas9 into a nickase is a D10A(aspartate to alanine at position 10 of Cas9) mutation in the RuvCdomain of Cas9 from S. pyogenes. Likewise, H939A (histidine to alanineat amino acid position 839) or H840A (histidine to alanine at amino acidposition 840) in the HNH domain of Cas9 from S. pyogenes can convert theCas9 into a nickase. Other examples of mutations that convert Cas9 intoa nickase include the corresponding mutations to Cas9 from S.thermophilus. See, e.g., Sapranauskas et al. (2011) Nucleic AcidsResearch 39:9275-9282 and WO 2013/141680, each of which is hereinincorporated by reference in its entirety. Such mutations can begenerated using well-known methods such as site-directed mutagenesis,PCR-mediated mutagenesis, or total gene synthesis. Examples of othermutations creating nickases can be found, for example, inWO/2013/176772A1 and WO/2013/142578A1, each of which is hereinincorporated by reference.

Cas proteins can also be fusion proteins. For example, a Cas protein canbe fused to a cleavage domain, an epigenetic modification domain, atranscriptional activation domain, or a transcriptional repressordomain. See WO 2014/089290, incorporated herein by reference in itsentirety. Cas proteins can also be fused to a heterologous polypeptideproviding increased or decreased stability. The fused domain orheterologous polypeptide can be located at the N-terminus, theC-terminus, or internally within the Cas protein.

One example of a Cas fusion protein is a Cas protein fused to aheterologous polypeptide that provides for subcellular localization.Such sequences can include, for example, a nuclear localization signal(NLS) such as the SV40 NLS for targeting to the nucleus, a mitochondriallocalization signal for targeting to the mitochondria, an ER retentionsignal, and the like. See, e.g., Lange et al. (2007) J. Biol. Chem.282:5101-5105. Such subcellular localization signals can be located atthe N-terminus, the C-terminus, or anywhere within the Cas protein. AnNLS can comprise a stretch of basic amino acids, and can be amonopartite sequence or a bipartite sequence.

Cas proteins can also comprise a cell-penetrating domain. For example,the cell-penetrating domain can be derived from the HIV-1 TAT protein,the TLM cell-penetrating motif from human hepatitis B virus, MPG, Pep-1,VP22, a cell penetrating peptide from Herpes simplex virus, or apolyarginine peptide sequence. See, for example, WO 2014/089290, hereinincorporated by reference in its entirety. The cell-penetrating domaincan be located at the N-terminus, the C-terminus, or anywhere within theCas protein.

Cas proteins can also comprise a heterologous polypeptide for ease oftracking or purification, such as a fluorescent protein, a purificationtag, or an epitope tag. Examples of fluorescent proteins include greenfluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald,Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellowfluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP,ZsYellowl), blue fluorescent proteins (e.g. eBFP, eBFP2, Azurite,mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g.eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescentproteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1,DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2,eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent proteins(mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine,tdTomato), and any other suitable fluorescent protein. Examples of tagsinclude glutathione-S-transferase (GST), chitin binding protein (CBP),maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinitypurification (TAP) tag, myc, AcV5, AU1 , AUS, E, ECS, E2, FLAG,hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV,KT3, S, 51 , T7, V5, VSV-G, histidine (His), biotin carboxyl carrierprotein (BCCP), and calmodulin.

Cas proteins can be provided in any form. For example, a Cas protein canbe provided in the form of a protein, such as a Cas protein complexedwith a gRNA. Alternatively, a Cas protein can be provided in the form ofa nucleic acid encoding the Cas protein, such as an RNA (e.g., messengerRNA (mRNA)) or DNA. Optionally, the nucleic acid encoding the Casprotein can be codon optimized for efficient translation into protein ina particular cell or organism (i.e., a human cell). When a nucleic acidencoding the Cas protein is introduced into the cell, the Cas proteincan be transiently, conditionally, or constitutively expressed in thecell.

Nucleic acids encoding Cas proteins can be stably integrated in thegenome of the cell and operably linked to a promoter active in the cell.Alternatively, nucleic acids encoding Cas proteins can be operablylinked to a promoter in an expression construct. Expression constructsinclude any nucleic acid constructs capable of directing expression of agene or other nucleic acid sequence of interest (e.g., a Cas gene) andwhich can transfer such a nucleic acid sequence of interest to a targetcell. For example, the nucleic acid encoding the Cas protein can be in atargeting vector comprising a nucleic acid insert and/or a vectorcomprising a DNA encoding the gRNA. Alternatively, it can be in a vectoror a plasmid that is separate from the targeting vector comprising thenucleic acid insert and/or separate from the vector comprising the DNAencoding the gRNA. Promoters that can be used in an expression constructinclude, for example, promoters active in a human iPS cell or anon-pluripotent cell transformed to express a naïve state. Suchpromoters can be, for example, conditional promoters, induciblepromoters, constitutive promoters, or tissue-specific promoters.

A “guide RNA” or “gRNA” includes an RNA molecule that binds to a Casprotein and targets the Cas protein to a specific location within atarget DNA. Guide RNAs can comprise two segments: a “DNA-targetingsegment” and a “protein-binding segment.” “Segment” includes a segment,section, or region of a molecule, such as a contiguous stretch ofnucleotides in an RNA. Some gRNAs comprise two separate RNA molecules:an “activator-RNA” and a “targeter-RNA.” Other gRNAs are a single RNAmolecule (single RNA polynucleotide), which can also be called a“single-molecule gRNA,” a “single-guide RNA,” or an “sgRNA.” See, e.g.,WO/2013/176772A1, WO/2014/065596A1, WO/2014/089290A1, WO/2014/093622A2,WO/2014/099750A2, WO/2013142578A1, and WO 2014/131833A1, each of whichis herein incorporated by reference. The terms “guide RNA” and “gRNA”are inclusive, including both double-molecule gRNAs and single-moleculegRNAs.

An exemplary two-molecule gRNA comprises a crRNA-like (“CRISPR RNA” or“targeter-RNA” or “crRNA” or “crRNA repeat”) molecule and acorresponding tracrRNA-like (“trans-acting CRISPR RNA” or“activator-RNA” or “tracrRNA” or “scaffold”) molecule. A crRNA comprisesboth the DNA-targeting segment (single-stranded) of the gRNA and astretch of nucleotides that forms one half of the dsRNA duplex of theprotein-binding segment of the gRNA.

A corresponding tracrRNA (activator-RNA) comprises a stretch ofnucleotides that forms the other half of the dsRNA duplex of theprotein-binding segment of the gRNA. A stretch of nucleotides of a crRNAare complementary to and hybridize with a stretch of nucleotides of atracrRNA to form the dsRNA duplex of the protein-binding domain of thegRNA. As such, each crRNA can be said to have a corresponding tracrRNA.

The crRNA and the corresponding tracrRNA hybridize to form a gRNA. ThecrRNA additionally provides the single stranded DNA-targeting segmentthat hybridizes to a target sequence. If used for modification within acell, the exact sequence of a given crRNA or tracrRNA molecule can bedesigned to be specific to the species in which the RNA molecules willbe used. See, for example, Mali et al. (2013) Science 339:823-826; Jineket al. (2012) Science 337:816-821; Hwang et al. (2013) Nat. Biotechnol.31:227-229; Jiang et al. (2013) Nat. Biotechnol. 31:233-239; and Cong etal. (2013) Science 339:819-823, each of which is herein incorporated byreference.

The DNA-targeting segment (crRNA) of a given gRNA comprises a nucleotidesequence that is complementary to a sequence in a target DNA. TheDNA-targeting segment of a gRNA interacts with a target DNA in asequence-specific manner via hybridization (i.e., base pairing). Assuch, the nucleotide sequence of the DNA-targeting segment may vary anddetermines the location within the target DNA with which the gRNA andthe target DNA will interact. The DNA-targeting segment of a subjectgRNA can be modified to hybridize to any desired sequence within atarget DNA. Naturally occurring crRNAs differ depending on the Cas9system and organism but often contain a targeting segment of between 21to 72 nucleotides length, flanked by two direct repeats (DR) of a lengthof between 21 to 46 nucleotides (see, e.g., WO2014/131833). In the caseof S. pyogenes, the DRs are 36 nucleotides long and the targetingsegment is 30 nucleotides long. The 3′ located DR is complementary toand hybridizes with the corresponding tracrRNA, which in turn binds tothe Cas9 protein.

The DNA-targeting segment can have a length of from about 12 nucleotidesto about 100 nucleotides. For example, the DNA-targeting segment canhave a length of from about 12 nucleotides (nt) to about 80 nt, fromabout 12 nt to about 50 nt, from about 12 nt to about 40 nt, from about12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 ntto about 20 nt, or from about 12 nt to about 19 nt. Alternatively, theDNA-targeting segment can have a length of from about 19 nt to about 20nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt,from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, fromabout 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about19 nt to about 60 nt, from about 19 nt to about 70 nt, from about 19 ntto about 80 nt, from about 19 nt to about 90 nt, from about 19 nt toabout 100 nt, from about 20 nt to about 25 nt, from about 20 nt to about30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt,from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, fromabout 20 nt to about 60 nt, from about 20 nt to about 70 nt, from about20 nt to about 80 nt, from about 20 nt to about 90 nt, or from about 20nt to about 100 nt.

The nucleotide sequence of the DNA-targeting segment that iscomplementary to a nucleotide sequence (target sequence) of the targetDNA can have a length at least about 12 nt. For example, theDNA-targeting sequence (i.e., the sequence within the DNA-targetingsegment that is complementary to a target sequence within the targetDNA) can have a length at least about 12 nt, at least about 15 nt, atleast about 18 nt, at least about 19 nt, at least about 20 nt, at leastabout 25 nt, at least about 30 nt, at least about 35 nt, or at leastabout 40 nt. Alternatively, the DNA-targeting sequence can have a lengthof from about 12 nucleotides (nt) to about 80 nt, from about 12 nt toabout 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt,from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, fromabout 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 ntto about 35 nt, from about 19 nt to about 40 nt, from about 19 nt toabout 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt,from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, fromabout 20 nt to about 45 nt, from about 20 nt to about 50 nt, or fromabout 20 nt to about 60 nt. In some cases, the DNA-targeting sequencecan have a length of at about 20 nt.

TracrRNAs can be in any form (e.g., full-length tracrRNAs or activepartial tracrRNAs) and of varying lengths. They can include primarytranscripts or processed forms. For example, tracrRNAs (as part of asingle-guide RNA or as a separate molecule as part of a two-moleculegRNA) may comprise or consist of all or a portion of a wild-typetracrRNA sequence (e.g., about or more than about 20, 26, 32, 45, 48,54, 63, 67, 85, or more nucleotides of a wild-type tracrRNA sequence).Examples of wild-type tracrRNA sequences from S. pyogenes include171-nucleotide, 89-nucleotide, 75-nucleotide, and 65-nucleotideversions. See, for example, Deltcheva et al. (2011) Nature 471:602-607;WO 2014/093661, each of which is incorporated herein by reference intheir entirety. Examples of tracrRNAs within single-guide RNAs (sgRNAs)include the tracrRNA segments found within +48, +54, +67, and +85versions of sgRNAs, where “+n” indicates that up to the +n nucleotide ofwild-type tracrRNA is included in the sgRNA. See U.S. Pat. No.8,697,359, incorporated herein by reference in its entirety.

The percent complementarity between the DNA-targeting sequence and thetarget sequence within the target DNA can be at least 60% (e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 98%, at least 99%, or100%). In some cases, the percent complementarity between theDNA-targeting sequence and the target sequence within the target DNA isat least 60% over about 20 contiguous nucleotides. In one example, thepercent complementarity between the DNA-targeting sequence and thetarget sequence within the target DNA is 100% over the 14 contiguousnucleotides at the 5′ end of the target sequence within thecomplementary strand of the target DNA and as low as 0% over theremainder. In such a case, the DNA-targeting sequence can be consideredto be 14 nucleotides in length. In another example, the percentcomplementarity between the DNA-targeting sequence and the targetsequence within the target DNA is 100% over the seven contiguousnucleotides at the 5′ end of the target sequence within thecomplementary strand of the target DNA and as low as 0% over theremainder. In such a case, the DNA-targeting sequence can be consideredto be 7 nucleotides in length.

The protein-binding segment of a gRNA can comprise two stretches ofnucleotides that are complementary to one another. The complementarynucleotides of the protein-binding segment hybridize to form a doublestranded RNA duplex (dsRNA). The protein-binding segment of a subjectgRNA interacts with a Cas protein, and the gRNA directs the bound Casprotein to a specific nucleotide sequence within target DNA via theDNA-targeting segment.

Guide RNAs can include modifications or sequences that provide foradditional desirable features (e.g., modified or regulated stability;subcellular targeting; tracking with a fluorescent label; a binding sitefor a protein or protein complex; and the like). Examples of suchmodifications include, for example, a 5′ cap (e.g., a 7-methylguanylatecap (m7G)); a 3′ polyadenylated tail (i.e., a 3′ poly(A) tail); ariboswitch sequence (e.g., to allow for regulated stability and/orregulated accessibility by proteins and/or protein complexes); astability control sequence; a sequence that forms a dsRNA duplex (i.e.,a hairpin)); a modification or sequence that targets the RNA to asubcellular location (e.g., nucleus, mitochondria, chloroplasts, and thelike); a modification or sequence that provides for tracking (e.g.,direct conjugation to a fluorescent molecule, conjugation to a moietythat facilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.); a modification or sequence that provides abinding site for proteins (e.g., proteins that act on DNA, includingtranscriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like); and combinations thereof.

Guide RNAs can be provided in any form. For example, the gRNA can beprovided in the form of RNA, either as two molecules (separate crRNA andtracrRNA) or as one molecule (sgRNA), and optionally in the form of acomplex with a Cas protein. The gRNA can also be provided in the form ofDNA encoding the RNA. The DNA encoding the gRNA can encode a single RNAmolecule (sgRNA) or separate RNA molecules (e.g., separate crRNA andtracrRNA). In the latter case, the DNA encoding the gRNA can be providedas separate DNA molecules encoding the crRNA and tracrRNA, respectively.

When a DNA encoding a gRNA is introduced into the cell, the gRNA can betransiently, conditionally, or constitutively expressed in the cell.DNAs encoding gRNAs can be stably integrated in the genome of the celland operably linked to a promoter active in the cell. Alternatively,DNAs encoding gRNAs can be operably linked to a promoter in anexpression construct. For example, the DNA encoding the gRNA can be in atargeting vector comprising a nucleic acid insert and/or a vectorcomprising a nucleic acid encoding a Cas protein. Alternatively, it canbe in a vector or a plasmid that is separate from the targeting vectorcomprising the nucleic acid insert and/or separate from the vectorcomprising the nucleic acid encoding the Cas protein. Such promoters canbe active, for example, in a human iPS cell or a non-pluripotent celltransformed to express a pluripotent state. Such promoters can be, forexample, conditional promoters, inducible promoters, constitutivepromoters, or tissue-specific promoters. In some instances, the promoteris an RNA polymerase III promoter, such as a human U6 promoter.

Alternatively, gRNAs can be prepared by various other methods. Forexample, gRNAs can be prepared by in vitro transcription using, forexample, T7 RNA polymerase (see, for example, WO 2014/089290 and WO2014/065596). Guide RNAs can also be a synthetically produced moleculeprepared by chemical synthesis.

A target sequence for a CRISPR/Cas system includes nucleic acidsequences present in a target DNA to which a DNA-targeting segment of agRNA will bind, provided sufficient conditions for binding exist. Forexample, target sequences include sequences to which a guide RNA isdesigned to have complementarity, where hybridization between a targetsequence and a DNA targeting sequence promotes the formation of a CRISPRcomplex. Full complementarity is not necessarily required, providedthere is sufficient complementarity to cause hybridization and promoteformation of a CRISPR complex. Target sequences also include cleavagesites for Cas proteins, described in more detail below. A targetsequence can comprise any polynucleotide, which can be located, forexample, in the nucleus or cytoplasm of a cell or within an organelle ofa cell, such as a mitochondrion or chloroplast.

The target sequence within a target DNA can be targeted by (i.e., bebound by, or hybridize with, or be complementary to) a Cas protein or agRNA. Suitable DNA/RNA binding conditions include physiologicalconditions normally present in a cell. Other suitable DNA/RNA bindingconditions (e.g., conditions in a cell-free system) are known in the art(see, e.g., Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook etal., Harbor Laboratory Press 2001)). The strand of the target DNA thatis complementary to and hybridizes with the Cas protein or gRNA can becalled the “complementary strand,” and the strand of the target DNA thatis complementary to the “complementary strand” (and is therefore notcomplementary to the Cas protein or gRNA) can be called“noncomplementary strand” or “template strand.”

The Cas protein can cleave the nucleic acid at a site within or outsideof a nucleic acid sequence present in a target DNA to which aDNA-targeting segment of a gRNA will bind. The “cleavage site” includesthe position of a nucleic acid at which a Cas protein produces asingle-strand break or a double-strand break. For example, formation ofa CRISPR complex (comprising a gRNA hybridized to a target sequence andcomplexed with a Cas protein) can result in cleavage of one or bothstrands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50,or more base pairs from) the nucleic acid sequence present in a targetDNA to which a DNA-targeting segment of a gRNA will bind. If thecleavage site is outside of the nucleic acid sequence present in atarget DNA to which a DNA-targeting segment of a gRNA will bind, thecleavage site is still considered to be within the “target sequence.”The cleavage site can be on only one strand or on both strands of anucleic acid. Cleavage sites can be at the same position on both strandsof the nucleic acid (producing blunt ends) or can be at different siteson each strand (producing staggered ends). Staggered ends can beproduced, for example, by using two Cas proteins which produce asingle-strand break at different cleavage sites on each strand. Forexample, a first nickase can create a single strand break on the firststrand of double stranded DNA (dsDNA), while a second nickase can createa single strand break on the second strand of dsDNA such thatoverhanging sequences are created. In some cases, the target sequence ofthe nickase on the first strand is separated from the target sequence ofthe nickase on the second strand by at least 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 40, 50, 75, 100, 250, 500, or 1,000 base pairs.

Site-specific cleavage of target DNA by Cas9 can occur at locationsdetermined by both (i) base-pairing complementarity between the gRNA andthe target DNA and (ii) a short motif, called the protospacer adjacentmotif (PAM), in the target DNA. The PAM can flank the target sequence.Optionally, the target sequence can be flanked on the 3′ end by the PAM.For example, the cleavage site of Cas9 can be about 1 to about 10 orabout 2 to about 5 base pairs (e.g., 3 base pairs) upstream ordownstream of the PAM sequence. In some cases (e.g., when Cas9 from S.pyogenes or a closely related Cas9 is used), the PAM sequence of thenon-complementary strand can be 5′-XGG-3′, where X is any DNA nucleotideand is immediately 3′ of the target sequence of the non-complementarystrand of the target DNA. As such, the PAM sequence of the complementarystrand would be 5′-CCY-3′, where Y is any DNA nucleotide and isimmediately 5′ of the target sequence of the complementary strand of thetarget DNA. In some such cases, X and Y can be complementary and the X-Ybase pair can be any base pair (e.g., X=C and Y=G; X=G and Y=C; X=A andY=T, X=T, and Y=A).

Examples of target sequences include a DNA sequence complementary to theDNA-targeting segment of a gRNA, or such a DNA sequence in addition to aPAM sequence. One example of a target sequence comprises the nucleotidesequence of GNNNNNNNNNNNNNNNNNNNNGG (GN₁₋₂₀GG; SEQ ID NO: 2). Othertarget sequences can have between 4-22 nucleotides in length of SEQ IDNO: 2, including the 5′ G and the 3′ GG. Yet other target sequences canhave between 14 and 20 nucleotides in length of SEQ ID NO: 2.

The target sequence can be any nucleic acid sequence endogenous orexogenous to a cell. The target sequence can be a sequence coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., a regulatorysequence or junk DNA) or can include both.

Active variants and fragments of nuclease agents (i.e. an engineerednuclease agent) are also provided. Such active variants can comprise atleast 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to the native nuclease agent, whereinthe active variants retain the ability to cut at a desired recognitionsite and hence retain nick or double-strand-break-inducing activity. Forexample, any of the nuclease agents described herein can be modifiedfrom a native endonuclease sequence and designed to recognize and inducea nick or double-strand break at a recognition site that was notrecognized by the native nuclease agent. Thus, in some embodiments, theengineered nuclease has a specificity to induce a nick or double-strandbreak at a recognition site that is different from the correspondingnative nuclease agent recognition site. Assays for nick ordouble-strand-break-inducing activity are known and generally measurethe overall activity and specificity of the endonuclease on DNAsubstrates containing the recognition site.

The nuclease agent may be introduced into the pluripotent cell by anymeans known in the art. The polypeptide encoding the nuclease agent maybe directly introduced into the cell. Alternatively, a polynucleotideencoding the nuclease agent can be introduced into the cell. When apolynucleotide encoding the nuclease agent is introduced into the cell,the nuclease agent can be transiently, conditionally or constitutivelyexpressed within the cell. Thus, the polynucleotide encoding thenuclease agent can be contained in an expression cassette and beoperably linked to a conditional promoter, an inducible promoter, aconstitutive promoter, or a tissue-specific promoter. Alternatively, thenuclease agent is introduced into the cell as an mRNA encoding anuclease agent.

In specific embodiments, the polynucleotide encoding the nuclease agentis stably integrated in the genome of the pluripotent cell and operablylinked to a promoter active in the cell. In other embodiments, thepolynucleotide encoding the nuclease agent is in the same targetingvector comprising the nucleic acid insert, while in other instances thepolynucleotide encoding the nuclease agent is in a vector or a plasmidthat is separate from the targeting vector comprising the nucleic acidinsert.

When the nuclease agent is provided to the pluripotent cell through theintroduction of a polynucleotide encoding the nuclease agent, such apolynucleotide encoding a nuclease agent can be modified to substitutecodons having a higher frequency of usage in the cell of interest, ascompared to the naturally occurring polynucleotide sequence encoding thenuclease agent. For example the polynucleotide encoding the nucleaseagent can be modified to substitute codons having a higher frequency ofusage in a human cell, as compared to the naturally occurringpolynucleotide sequence.

b. Selection Markers

Various selection markers can be used in the methods and compositionsdisclosed herein which provide for modifying a target genomic locus on achromosome. Such markers are disclosed elsewhere herein and include, butare not limited to, selection markers that impart resistance to anantibiotic such as G418, hygromycin, blasticidin, neomycin, orpuromycin. The polynucleotide encoding the selection markers areoperably linked to a promoter active in a human iPS cell or anon-pluripotent cell transformed to express a naïve state.

c. Target Genomic Locus

Various methods and compositions are provided which allow for theintegration of at least one nucleic acid insert at a target genomiclocus on a chromosome. A “target genomic locus on a chromosome”comprises any segment or region of DNA on a chromosome that one desiresto integrate a nucleic acid insert. The genomic locus on a chromosomebeing targeted can be native to human iPS cell or a non-pluripotent celltransformed to express a pluripotent state, or alternatively cancomprise a heterologous or exogenous segment of DNA that was integratedinto a chromosome of the cell. Such heterologous or exogenous segmentsof DNA can include transgenes, expression cassettes, polynucleotideencoding selection makers, or heterologous or exogenous regions ofgenomic DNA. The target genomic locus on the chromosome can comprise anyof the targeted genomic integration system including, for example, therecognition site, the selection marker, previously integrated nucleicacid inserts, polynucleotides encoding nuclease agents, promoters, etc.Alternatively, the target genomic locus on the chromosome can be locatedwithin a yeast artificial chromosome (YAC), bacterial artificialchromosome (BAC), a human artificial chromosome, or any other engineeredgenomic region contained in an appropriate host cell. Thus, in specificembodiments, the targeted genomic locus on the chromosome can comprisenative genomic sequence from a human cell or heterologous or exogenousgenomic nucleic acid sequence from a non-human mammal, a non-human cell,a rodent, a human, a rat, a mouse, a hamster, a rabbit, a pig, a bovine,a deer, a sheep, a goat, a chicken, a cat, a dog, a ferret, a primate(e.g., marmoset, rhesus monkey), domesticated mammal or an agriculturalmammal, or any other organism of interest or a combination thereof.

d. Targeting Vectors and Nucleic Acid Inserts

As outlined above, methods and compositions provided herein employtargeting vectors alone or in combination with a nuclease agent.“Homologous recombination” is used conventionally to refer to theexchange of DNA fragments between two DNA molecules at cross-over siteswithin the regions of homology.

i. Nucleic Acid Insert

One or more separate nucleic acid inserts can be employed in the methodsdisclosed herein, and they can be introduced into a cell via separatetargeting vectors or on the same targeting vector. Nucleic acid insertsinclude segments of DNA to be integrated at genomic target loci.Integration of a nucleic acid insert at a target locus can result inaddition of a nucleic acid sequence of interest to the target locus,deletion of a nucleic acid sequence of interest at the target locus,and/or replacement of a nucleic acid sequence of interest at the targetlocus.

The nucleic acid insert or the corresponding nucleic acid at the targetlocus being replaced can be a coding region, an intron, an exon, anuntranslated region, a regulatory region, a promoter, an enhancer, orany combination thereof. Moreover, the nucleic acid insert or thecorresponding nucleic acid at the target locus being replaced can be ofany desired length, including, for example, between 10-100 nucleotidesin length, 100-500 nucleotides in length, 500 nucleotides-1 kb inlength, 1 kb to 1.5 kb nucleotide in length, 1.5 kb to 2 kb nucleotidesin length, 2 kb to 2.5 kb nucleotides in length, 2.5 kb to 3 kbnucleotides in length, 3 kb to 5 kb nucleotides in length, 5 kb to 8 kbnucleotides in length, 8 kb to 10 kb nucleotides in length or more. Inother cases, the length can be from about 5 kb to about 10 kb, fromabout 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kbto about 100 kb, from about 100 kb to about 150 kb, from about 150 kb toabout 200 kb, from about 200 kb to about 250 kb, from about 250 kb toabout 300 kb, from about 300 kb to about 350 kb, from about 350 kb toabout 400 kb, from about 400 kb to about 800 kb, from about 800 kb to 1Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb,from about 2 Mb, to about 2.5 Mb, from about 2.5 Mb to about 2.8 Mb,from about 2.8 Mb to about 3 Mb. In yet other cases, the length can beat least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides orat least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb or greater.

In some targeting vectors, the nucleic acid insert can be from about 5kb to about 200 kb, from about 5 kb to about 10 kb, from about 10 kb toabout 20 kb, from about 20 kb to about 30 kb, from about 30 kb to about40 kb, from about 40 kb to about 50 kb, from about 60 kb to about 70 kb,from about 80 kb to about 90 kb, from about 90 kb to about 100 kb, fromabout 100 kb to about 110 kb, from about 120 kb to about 130 kb, fromabout 130 kb to about 140 kb, from about 140 kb to about 150 kb, fromabout 150 kb to about 160 kb, from about 160 kb to about 170 kb, fromabout 170 kb to about 180 kb, from about 180 kb to about 190 kb, fromabout 190 kb to about 200 kb. Alternatively, the nucleic acid insert canbe from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, fromabout 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100kb to about 150 kb, from about 150 kb to about 200 kb, from about 200 kbto about 250 kb, from about 250 kb to about 300 kb, from about 300 kb toabout 350 kb, or from about 350 kb to about 400 kb.

In some cases, the replacement of the nucleic acid at the target locusresults in the deletion of a target sequence ranging from about 1 kb toabout 200 kb, from about 2 kb to about 20 kb, or from about 0.5 kb toabout 3 Mb. In some cases, the extent of the deletion is greater than atotal length of the 5′ homology arm and the 3′ homology arm.

In some cases, the extent of the deletion of the target sequence rangesfrom about 5 kb to about 10 kb, from about 10 kb to about 20 kb, fromabout 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100kb to about 150 kb, from about 150 kb to about 200 kb, from about 20 kbto about 30 kb, from about 30 kb to about 40 kb, from about 40 kb toabout 50 kb, from about 50 kb to about 60 kb, from about 60 kb to about70 kb, from about 70 kb to about 80 kb, from about 80 kb to about 90 kb,from about 90 kb to about 100 kb, from about 100 kb to about 110 kb,from about 110 kb to about 120 kb, from about 120 kb to about 130 kb,from about 130 kb to about 140 kb, from about 140 kb to about 150 kb,from about 150 kb to about 160 kb, from about 160 kb to about 170 kb,from about 170 kb to about 180 kb, from about 180 kb to about 190 kb,from about 190 kb to about 200 kb, from about 200 kb to about 250 kb,from about 250 kb to about 300 kb, from about 300 kb to about 350 kb,from about 350 kb to about 400 kb, from about 400 kb to about 800 kb,from about 800 kb to 1 Mb, from about 1 Mb to about 1.5 Mb, from about1.5 Mb to about 2 Mb, from about 2 Mb, to about 2.5 Mb, from about 2.5Mb to about 2.8 Mb, from about 2.8 Mb to about 3 Mb, from about 200 kbto about 300 kb, from about 300 kb to about 400 kb, from about 400 kb toabout 500 kb, from about 500 kb to about 1 Mb, from about 1 Mb to about1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5Mb, or from about 2.5 Mb to about 3 Mb.

In other cases, the nucleic acid insert or the corresponding nucleicacid at the target locus being replaced can be at least 10 kb, at least20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb,at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, atleast 150 kb, at least 200 kb, at least 250 kb, at least 300 kb, atleast 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb orgreater.

The nucleic acid insert can comprise genomic DNA or any other type ofDNA. For example, the nucleic acid insert can be from a prokaryote, aeukaryote, a yeast, a bird (e.g., chicken), a non-human mammal, arodent, a human, a rat, a mouse, a hamster a rabbit, a pig, a bovine, adeer, a sheep, a goat, a cat, a dog, a ferret, a primate (e.g.,marmoset, rhesus monkey), a domesticated mammal, an agricultural mammal,or any other organism of interest. In one example, the insertpolynucleotide can comprise any human or non-human genomic locus.

The nucleic acid insert and/or the nucleic acid at the target locus cancomprise a coding sequence or a non-coding sequence, such as aregulatory element (e.g., a promoter, an enhancer, or a transcriptionalrepressor-binding element). For example, the nucleic acid insert cancomprise a knock-in allele of at least one exon of an endogenous gene,or a knock-in allele of the entire endogenous gene (i.e., “gene-swapknock-in”).

The nucleic acid insert can also comprise a conditional allele. Theconditional allele can be a multifunctional allele, as described in US2011/0104799, which is incorporated by reference in its entirety. Forexample, the conditional allele can comprise: (a) an actuating sequencein sense orientation with respect to transcription of a target gene; (b)a drug selection cassette (DSC) in sense or antisense orientation; (c) anucleotide sequence of interest (NSI) in antisense orientation; and (d)a conditional by inversion module (COIN, which utilizes anexon-splitting intron and an invertible gene-trap-like module) inreverse orientation. See, for example, US 2011/0104799, which isincorporated by reference in its entirety. The conditional allele canfurther comprise recombinable units that recombine upon exposure to afirst recombinase to form a conditional allele that (i) lacks theactuating sequence and the DSC; and (ii) contains the NSI in senseorientation and the COIN in antisense orientation. See US 2011/0104799.

Some nucleic acid inserts comprise a polynucleotide encoding a selectionmarker. The selection marker can be contained in a selection cassette.Such selection markers include, but are not limited, to neomycinphosphotransferase (neon), hygromycin B phosphotransferase (hygr),puromycin-N-acetyltransferase (puror), blasticidin S deaminase (bsrr),xanthine/guanine phosphoribosyl transferase (gpt), or herpes simplexvirus thymidine kinase (HSV-k), or a combination thereof. Thepolynucleotide encoding the selection marker can be operably linked to apromoter active in a cell being targeted.

In some targeting vectors, the nucleic acid insert comprises a reportergene. Examples of reporter genes are genes encoding luciferase,β-galactosidase, green fluorescent protein (GFP), enhanced greenfluorescent protein (eGFP), cyan fluorescent protein (CFP), yellowfluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP),blue fluorescent protein (BFP), enhanced blue fluorescent protein(eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry,J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean,T-Sapphire, alkaline phosphatase, and a combination thereof. Suchreporter genes can be operably linked to a promoter active in a cellbeing targeted.

In some targeting vectors, the nucleic acid insert comprises one or moreexpression cassettes or deletion cassettes. A given cassette cancomprise a nucleotide sequence of interest, a nucleic acid encoding aselection marker, and/or a reporter gene, along with various regulatorycomponents that influence expression. Examples of selectable markers andreporter genes that can be included are discussed in detail elsewhereherein.

In some targeting vectors, the insert nucleic acid comprises a nucleicacid flanked with site-specific recombination target sequences. Althoughthe entire insert nucleic acid can be flanked by such site-specificrecombination target sequences, any region or individual polynucleotideof interest within the insert nucleic acid can also be flanked by suchsites. Site-specific recombination target sequences, which can flank theinsert nucleic acid or any polynucleotide of interest in the insertnucleic acid can include, for example, loxP, lox511, lox2272, lox66,lox71, loxM2, lox5171, FRT, FRT11, FRT71, attp, att, FRT, rox, and acombination thereof. In one example, the site-specific recombinationsites flank a polynucleotide encoding a selection marker and/or areporter gene contained within the insert nucleic acid. Followingintegration of the insert nucleic acid at a targeted locus, thesequences between the site-specific recombination sites can be removed.

ii. Targeting Vectors

Targeting vectors can be employed to introduce the nucleic acid insertinto a target genomic locus and comprise the nucleic acid insert andhomology arms that flank the nucleic acid insert. Targeting vectors canbe in linear form or in circular form, and can be single-stranded ordouble-stranded. For ease of reference, the homology arms are referredto herein as 5′ and 3′ (i.e., upstream and downstream) homology arms.This terminology relates to the relative position of the homology armsto the nucleic acid insert within the targeting vector. The 5′ and 3′homology arms correspond to regions within the targeted locus, which arereferred to herein as “5′ target sequence” and “3′ target sequence,”respectively.

A homology arm and a target sequence “correspond” or are “corresponding”to one another when the two regions share a sufficient level of sequenceidentity to one another to act as substrates for a homologousrecombination reaction. The term “homology” includes DNA sequences thatare either identical or share sequence identity to a correspondingsequence. The sequence identity between a given target sequence and thecorresponding homology arm found on the targeting vector can be anydegree of sequence identity that allows for homologous recombination tooccur. For example, the amount of sequence identity shared by thehomology arm of the targeting vector (or a fragment thereof) and thetarget sequence (or a fragment thereof) can be at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity,such that the sequences undergo homologous recombination. Moreover, acorresponding region of homology between the homology arm and thecorresponding target sequence can be of any length that is sufficient topromote homologous recombination at the cleaved recognition site. Forexample, a given homology arm and/or corresponding target sequence cancomprise corresponding regions of homology that are at least about 5-10kb, 5-15 kb, 5-20 kb, 5-25 kb, 5-30 kb, 5-35 kb, 5-40 kb, 5-45 kb, 5-50kb, 5-55 kb, 5-60 kb, 5-65 kb, 5-70 kb, 5-75 kb, 5-80 kb, 5-85 kb, 5-90kb, 5-95 kb, 5-100 kb, 100-200 kb, or 200-300 kb in length or more (suchas described in the LTVEC vectors described elsewhere herein) such thatthe homology arm has sufficient homology to undergo homologousrecombination with the corresponding target sequences within the genomeof the cell.

The homology arms can correspond to a locus that is native to a cell(e.g., the targeted locus), or alternatively they can correspond to aregion of a heterologous or exogenous segment of DNA that was integratedinto the genome of the cell, including, for example, transgenes,expression cassettes, or heterologous or exogenous regions of DNA.Alternatively, the homology arms of the targeting vector can correspondto a region of a yeast artificial chromosome (YAC), a bacterialartificial chromosome (BAC), a human artificial chromosome, or any otherengineered region contained in an appropriate host cell. Still further,the homology arms of the targeting vector can correspond to or bederived from a region of a BAC library, a cosmid library, or a P1 phagelibrary. In certain instances, the homology arms of the targeting vectorcorrespond to a locus that is native, heterologous, or exogenous to ahuman iPS cell or a non-pluripotent cell transformed to express a naïvestate. In some cases, the homology arms correspond to a locus of thecell that is not targetable using a conventional method or that can betargeted only incorrectly or only with significantly low efficiency inthe absence of a nick or double-strand break induced by a nuclease agent(e.g., a Cas protein). In some cases, the homology arms are derived fromsynthetic DNA.

In some targeting vectors, the 5′ and 3′ homology arms correspond to asafe harbor locus. Interactions between integrated exogenous DNA and ahost genome can limit the reliability and safety of integration and canlead to overt phenotypic effects that are not due to the targetedgenetic modification but are instead due to unintended effects of theintegration on surrounding endogenous genes. For example, randomlyinserted transgenes can be subject to position effects and silencing,making their expression unreliable and unpredictable. Likewise,integration of exogenous DNA into a chromosomal locus can affectsurrounding endogenous genes and chromatin, thereby altering cellbehavior and phenotypes. Safe harbor loci include chromosomal loci wheretransgenes or other exogenous nucleic acid inserts can be stably andreliably expressed in all tissues of interest without overtly alteringcell behavior or phenotype. See, e.g., Sadelain et al. (2012) Nat. Rev.Cancer 12:51-58. For example, safe harbor loci can include chromosomalloci where exogenous DNA can integrate and function in a predictablemanner without adversely affecting endogenous gene structure orexpression. Safe harbor loci can include extragenic regions orintragenic regions such as, for example, loci within genes that arenon-essential, dispensable, or able to be disrupted without overtphenotypic consequences.

For example, the Rosa26 locus and its equivalent in humans offer an openchromatin configuration in all tissues and is ubiquitously expressedduring embryonic development and in adults. See Zambrowicz et al. (1997)Proc. Natl. Acad. Sci. USA 94:3789-3794. In addition, the Rosa26 locuscan be targeted with high efficiency, and disruption of the Rosa26 geneproduces no overt phenotype. Another example of a suitable locus is theCh25h locus.

A homology arm of a targeting vector can be of any length that issufficient to promote a homologous recombination event with acorresponding target sequence, including, for example, at least 5-10 kb,5-15 kb, 5-20 kb, 5-25 kb, 5-30 kb, 5-35 kb, 5-40 kb, 5-45 kb, 5-50 kb,5-55 kb, 5-60 kb, 5-65 kb, 5-70 kb, 5-75 kb, 5-80 kb, 5-85 kb, 5-90 kb,5-95 kb, 5-100 kb, 100- 200 kb, or 200-300 kb in length or greater. Asdescribed in further detail below, large targeting vectors can employtargeting arms of greater length.

Nuclease agents (e.g., CRISPR/Cas systems) can be employed incombination with targeting vectors to aid in the modification of atarget locus. Such nuclease agents may promote homologous recombinationbetween the targeting vector and the target locus. When nuclease agentsare employed in combination with a targeting vector, the targetingvector can comprise 5′ and 3′ homology arms corresponding to 5′ and 3′target sequences located in sufficient proximity to a nuclease cleavagesite so as to promote the occurrence of a homologous recombination eventbetween the target sequences and the homology arms upon a nick ordouble-strand break at the nuclease cleavage site. The term “nucleasecleavage site” includes a DNA sequence at which a nick or double-strandbreak is created by a nuclease agent (e.g., a Cas9 cleavage site). Thetarget sequences within the targeted locus that correspond to the 5′ and3′ homology arms of the targeting vector are “located in sufficientproximity” to a nuclease cleavage site if the distance is such as topromote the occurrence of a homologous recombination event between the5′ and 3′ target sequences and the homology arms upon a nick ordouble-strand break at the recognition site. Thus, in specificinstances, the target sequences corresponding to the 5′ and/or 3′homology arms of the targeting vector are within at least 1 nucleotideof a given recognition site or are within at least 10 nucleotides toabout 14 kb of a given recognition site. In some cases, the nucleasecleavage site is immediately adjacent to at least one or both of thetarget sequences.

The spatial relationship of the target sequences that correspond to thehomology arms of the targeting vector and the nuclease cleavage site canvary. For example, target sequences can be located 5′ to the nucleasecleavage site, target sequences can be located 3′ to the recognitionsite, or the target sequences can flank the nuclease cleavage site.

Combined use of the targeting vector (including, for example, a largetargeting vector) with a nuclease agent can result in an increasedtargeting efficiency compared to use of the targeting vector alone. Forexample, when a targeting vector is used in conjunction with a nucleaseagent, targeting efficiency of the targeting vector can be increased byat least two-fold, at least three-fold, at least 4-fold, or at least10-fold when compared to use of the targeting vector alone.

iii. Large Targeting Vectors

Some targeting vectors are “large targeting vectors” or “LTVECs,” whichincludes targeting vectors that comprise homology arms that correspondto and are derived from nucleic acid sequences larger than thosetypically used by other approaches intended to perform homologousrecombination in cells. Examples of generating targeted geneticmodifications using LTVECs are disclosed, for example, in WO2015/088643, US 2015/0159175, US 2015/0159174, US 2014/0310828, US2014/0309487, and US 2013-0309670, each of which is herein incorporatedby reference in its entirety for all purposes. LTVECs also includetargeting vectors comprising nucleic acid inserts having nucleic acidsequences larger than those typically used by other approaches intendedto perform homologous recombination in cells. For example, LTVECs makepossible the modification of large loci that cannot be accommodated bytraditional plasmid-based targeting vectors because of their sizelimitations. For example, the targeted locus can be (i.e., the 5′ and 3′homology arms can correspond to) a locus of the cell that is nottargetable using a conventional method or that can be targeted onlyincorrectly or only with significantly low efficiency in the absence ofa nick or double-strand break induced by a nuclease agent (e.g., a Casprotein).

Examples of LTVECs include vectors derived from a bacterial artificialchromosome (BAC), a human artificial chromosome, or a yeast artificialchromosome (YAC). Non-limiting examples of LTVECs and methods for makingthem are described, e.g., in U.S. Pat. No., 6,586,251; U.S. Pat. No.6,596,541; U.S. Pat. No. 7,105,348; and WO 2002/036789 (PCT/US01/45375),each of which is herein incorporated by reference. LTVECs can be inlinear form or in circular form.

LTVECs can be of any length, including, for example, from about 50 kb toabout 300 kb, from about 50 kb to about 75 kb, from about 75 kb to about100 kb, from about 100 kb to 125 kb, from about 125 kb to about 150 kb,from about 150 kb to about 175 kb, about 175 kb to about 200 kb, fromabout 200 kb to about 225 kb, from about 225 kb to about 250 kb, fromabout 250 kb to about 275 kb or from about 275 kb to about 300 kb.Alternatively, an LTVEC can be at least 10 kb, at least 15 kb, at least20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb,at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, atleast 150 kb, at least 200 kb, at least 250 kb, at least 300 kb, atleast 350 kb, at least 400 kb, at least 450 kb, or at least 500 kb orgreater. The size of an LTVEC can be too large to enable screening oftargeting events by conventional assays, e.g., southern blotting andlong-range (e.g., 1 kb to 5 kb) PCR.

In some cases, an LTVEC comprises a nucleic acid insert ranging fromabout 5 kb to about 200 kb, from about 5 kb to about 10 kb, from about10 kb to about 20 kb, from about 20 kb to about 30 kb, from about 30 kbto about 40 kb, from about 40 kb to about 50 kb, from about 60 kb toabout 70 kb, from about 80 kb to about 90 kb, from about 90 kb to about100 kb, from about 100 kb to about 110 kb, from about 120 kb to about130 kb, from about 130 kb to about 140 kb, from about 140 kb to about150 kb, from about 150 kb to about 160 kb, from about 160 kb to about170 kb, from about 170 kb to about 180 kb, from about 180 kb to about190 kb, or from about 190 kb to about 200 kb. In other cases, the insertnucleic acid can range from about 5 kb to about 10 kb, from about 10 kbto about 20 kb, from about 20 kb to about 40 kb, from about 40 kb toabout 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about100 kb, from about 100 kb to about 150 kb, from about 150 kb to about200 kb, from about 200 kb to about 250 kb, from about 250 kb to about300 kb, from about 300 kb to about 350 kb, or from about 350 kb to about400 kb.

In some LTVECs, the sum total of the upstream homology arm and thedownstream homology arm is at least 10 kb. In other LTVECs, the upstreamhomology arm ranges from about 5 kb to about 100 kb and/or thedownstream homology arm ranges from about 5 kb to about 100 kb. The sumtotal of the upstream and downstream homology arms can be, for example,from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, fromabout 20 kb to about 30 kb, from about 30 kb to about 40 kb, from about40 kb to about 50 kb, from about 50 kb to about 60 kb, from about 60 kbto about 70 kb, from about 70 kb to about 80 kb, from about 80 kb toabout 90 kb, from about 90 kb to about 100 kb, from about 100 kb toabout 110 kb, from about 110 kb to about 120 kb, from about 120 kb toabout 130 kb, from about 130 kb to about 140 kb, from about 140 kb toabout 150 kb, from about 150 kb to about 160 kb, from about 160 kb toabout 170 kb, from about 170 kb to about 180 kb, from about 180 kb toabout 190 kb, or from about 190 kb to about 200 kb.

In some cases, the LTVEC and nucleic acid insert are designed to allowfor a deletion at the target locus from about 5 kb to about 10 kb, fromabout 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kbto about 100 kb, from about 100 kb to about 150 kb, or from about 150 kbto about 200 kb, from about 200 kb to about 300 kb, from about 300 kb toabout 400 kb, from about 400 kb to about 500 kb, from about 500 kb toabout 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about2 Mb, from about 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3Mb. Alternatively, the deletion can be at least 10 kb, at least 20 kb,at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb,at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, atleast 400 kb, at least 450 kb, or at least 500 kb or greater.

In other cases, the LTVEC and nucleic acid insert are designed to allowfor an insertion into the target locus of an exogenous nucleic acidsequence ranging from about 5 kb to about 10 kb, from about 10 kb toabout 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100kb, from about 100 kb to about 150 kb, from about 150 kb to about 200kb, from about 200 kb to about 250 kb, from about 250 kb to about 300kb, from about 300 kb to about 350 kb, or from about 350 kb to about 400kb. Alternatively, the insertion can be at least 10 kb, at least 20 kb,at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb,at least 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, atleast 400 kb, at least 450 kb, or at least 500 kb or greater.

In yet other cases, the nucleic acid insert and/or the region of theendogenous locus being deleted is at least 100, 200, 300, 400, 500, 600,700, 800, or 900 nucleotides or at least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb orgreater.

iv. Methods of Integrating a Nucleic acid insert Near the RecognitionSite on a Chromosome by Homologous Recombination

In some examples, methods for modifying a target genomic locus on achromosome in a pluripotent cell can comprise: (a) providing a cellcomprising a target genomic locus on a chromosome, (b) introducing intothe cell a first targeting vector comprising a first nucleic acid insertflanked by 5′ and 3′ homology arms corresponding to 5′ and 3′ targetsequences; and (c) identifying at least one cell comprising in itsgenome the first nucleic acid insert integrated at the target genomiclocus on the chromosome. As discussed in detail elsewhere herein, inspecific embodiments, the sum total of the first homology arm and thesecond homology arm of the targeting vector is about 4 kb, 5 kb, 6 kb, 7kb, 8 kb, 9 kb, about 4 kb to about 5 kb, about 5 kb to about 6 kb,about 6 kb to about 7 kb, about 8 kb to about 9 kb, or is at least 10 kbor at least 10 kb and less than 150 kb. In specific embodiments, anLTVEC is employed. In one non-limiting embodiment, such methods areperformed employing the culture medium provided herein.

In other examples, methods for modifying a target genomic locus on achromosome in a pluripotent cell can comprise: (a) providing a cellcomprising a target genomic locus on a chromosome comprising arecognition site for a nuclease agent, (b) introducing into the cell (i)the nuclease agent, wherein the nuclease agent induces a nick ordouble-strand break at the first recognition site; and, (ii) a firsttargeting vector comprising a first nucleic acid insert flanked by 5′and 3′ homology arms corresponding to 5′ and 3′ target sequences locatedin sufficient proximity to the first recognition site; and (c)identifying at least one cell comprising in its genome the first nucleicacid insert integrated at the target genomic locus on the chromosome. Asdiscussed in detail elsewhere herein, in specific embodiments, the sumtotal of the first homology arm and the second homology arm of thetargeting vector is about 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, about 4 kbto about 5 kb, about 5 kb to about 6 kb, about 6 kb to about 7 kb, about8 kb to about 9 kb, or is at least 10 kb or at least 10 kb and less than150 kb. In specific embodiments, an LTVEC is employed. In onenon-limiting embodiment, such methods are performed employing theculture medium provided herein.

Various methods can also be employed to identify pluripotent cellshaving the nucleic acid insert integrated at the genomic target locus.Insertion of the nucleic acid insert at the genomic target locus resultsin a “modification of allele.” The term “modification of allele” or“MOA” includes the modification of the exact DNA sequence of one alleleof a gene(s) or chromosomal locus (loci) in a genome. Examples of“modification of allele (MOA)” include, but are not limited to,deletions, substitutions, or insertions of as little as a singlenucleotide or deletions of many kilobases spanning a gene(s) orchromosomal locus (loci) of interest, as well as any and all possiblemodifications between these two extremes.

In various embodiments, to facilitate identification of the targetedmodification, a high-throughput quantitative assay, namely, modificationof allele (MOA) assay, is employed. The MOA assay described hereinallows a large-scale screening of a modified allele(s) in a parentalchromosome following a genetic modification. The MOA assay can becarried out via various analytical techniques, including, but notlimited to, a quantitative PCR, e.g., a real-time PCR (qPCR). Forexample, the real-time PCR comprises a first primer-probe set thatrecognizes the target locus and a second primer-probe set thatrecognizes a non-targeted reference locus. In addition, the primer-probeset comprises a fluorescent probe that recognizes the amplifiedsequence. The quantitative assay can also be carried out via a varietyof analytical techniques, including, but not limited to,fluorescence-mediated in situ hybridization (FISH), comparative genomichybridization, isothermic DNA amplification, quantitative hybridizationto an immobilized probe(s), Invader Probes®, MMP assays®, TaqMan®Molecular Beacon, and Eclipse™ probe technology. See, for example,US2005/0144655, incorporated by reference herein in its entirety.

In various embodiments, in the presence of the nick or double strandbreak, targeting efficiency of a targeting vector (such as a LTVEC) atthe target genomic locus is at least about 2-fold higher, at least about3-fold higher, at least about 4-fold higher than in the absence of thenick or double-strand break (using, e.g., the same targeting vector andthe same homology arms and corresponding target sites at the genomiclocus of interest but in the absence of an added nuclease agent thatmakes the nick or double strand break).

The various methods set forth above can be sequentially repeated toallow for the targeted integration of any number of nucleic acid insertsinto a given targeted genomic locus on a chromosome. Thus, the variousmethods provide for the insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid insertsinto the target genomic locus on a chromosome. In particularembodiments, such sequential tiling methods allow for the reconstructionof large genomic regions from an animal cell or from a mammalian cell(i.e., a human, a non-human, a rodent, a mouse, a monkey, a rat, ahamster, a domesticated mammal or an agricultural animal) into atargeted genomic locus on a chromosome. In such instances, the transferand reconstruction of genomic regions that include both coding andnon-coding regions allow for the complexity of a given region to bepreserved by retaining, at least in part, the coding regions, thenon-coding regions and the copy number variations found within thenative genomic region. Thus, the various methods provide, for example,methods to generate “heterologous” or “exogenous” genomic regions withina human iPS cell or a non-pluripotent cell transformed to express apluripotent state.

v. Polynucleotides of Interest

Any polynucleotide of interest may be contained in the various nucleicacid inserts and thereby integrated at the target genomic locus on achromosome. The methods disclosed herein, provide for at least 1, 2, 3,4, 5, 6 or more polynucleotides of interest to be integrated into thetargeted genomic locus.

The polynucleotide of interest within the nucleic acid insert whenintegrated at the target genomic locus on a chromosome can introduce oneor more genetic modifications into the pluripotent cell. The geneticmodification can comprise a deletion of an endogenous nucleic acidsequence and/or the addition of an exogenous or heterologous ororthologous polynucleotide into the target genomic locus. In oneembodiment, the genetic modification comprises a replacement of anendogenous nucleic acid sequence with an exogenous polynucleotide ofinterest at the target genomic locus. Thus, methods provided hereinallow for the generation of a genetic modification comprising aknockout, a deletion, an insertion, a replacement (“knock-in”), a pointmutation, a domain swap, an exon swap, an intron swap, a regulatorysequence swap, a gene swap, or a combination thereof in a target genomiclocus on a chromosome. Such modifications may occur upon integration ofthe first, second, third, fourth, fifth, six, seventh, or any subsequentnucleic acid inserts into the target genomic locus.

The polynucleotide of interest within the nucleic acid insert and/orintegrated at the target genomic locus can comprise a sequence that isnative or homologous to the pluripotent cell it is introduced into; thepolynucleotide of interest can be heterologous to the cell it isintroduced to; the polynucleotide of interest can be exogenous to thecell it is introduced into; the polynucleotide of interest can beorthologous to the cell it is introduced into; or the polynucleotide ofinterest can be from a different species than the cell it is introducedinto. “Homologous” in reference to a sequence includes a sequence thatis native to the cell. “Heterologous” in reference to a sequenceincludes a sequence that originates from a foreign species, or, if fromthe same species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention.“Exogenous” in reference to a sequence includes a sequence thatoriginates from a foreign species. “Orthologous” includes apolynucleotide from one species that is functionally equivalent to aknown reference sequence in another species (i.e., a species variant).The polynucleotide of interest can be from any organism of interestincluding, but not limited to, non-human, a rodent, a hamster, a mouse,a rat, a human, a monkey, an avian, an agricultural mammal or anon-agricultural mammal The polynucleotide of interest can furthercomprise a coding region, a non-coding region, a regulatory region, or agenomic DNA. Thus, the 1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th),7^(th), and/or any of the subsequent nucleic acid inserts can comprisesuch sequences.

In one embodiment, the polynucleotide of interest within the nucleicacid insert and/or integrated at the target genomic locus on achromosome is homologous to a human nucleic acid. In still furtherembodiments, the polynucleotide of interest integrated at the targetlocus is a fragment of a genomic nucleic acid. In one embodiment, thegenomic nucleic acid is a mouse genomic nucleic acid, a human genomicnucleic acid, a non-human nucleic acid, a rodent nucleic acid, a ratnucleic acid, a hamster nucleic acid, a monkey nucleic acid, anagricultural mammal nucleic acid or a non-agricultural mammal nucleicacid or a combination thereof.

In one embodiment, the polynucleotide of interest can range from about500 nucleotides to about 200 kb as described above. The polynucleotideof interest can be from about 500 nucleotides to about 5 kb, from about5 kb to about 200 kb, from about 5 kb to about 10 kb, from about 10 kbto about 20 kb, from about 20 kb to about 30 kb, from about 30 kb toabout 40 kb, from about 40 kb to about 50 kb, from about 60 kb to about70 kb, from about 80 kb to about 90 kb, from about 90 kb to about 100kb, from about 100 kb to about 110 kb, from about 120 kb to about 130kb, from about 130 kb to about 140 kb, from about 140 kb to about 150kb, from about 150 kb to about 160 kb, from about 160 kb to about 170kb, from about 170 kb to about 180 kb, from about 180 kb to about 190kb, or from about 190 kb to about 200 kb.

The polynucleotide of interest within the nucleic acid insert and/orinserted at the target genomic locus on a chromosome can encode apolypeptide, can encode an miRNA, can encode a long non-coding RNA, orit can comprise any regulatory regions or non-coding regions of interestincluding, for example, a regulatory sequence, a promoter sequence, anenhancer sequence, a transcriptional repressor-binding sequence, or adeletion of a non-protein-coding sequence, but does not comprise adeletion of a protein-coding sequence. In addition, the polynucleotideof interest within the nucleic acid insert and/or inserted at the targetgenomic locus on a chromosome can encode a protein expressed in thenervous system, the skeletal system, the digestive system, thecirculatory system, the muscular system, the respiratory system, thecardiovascular system, the lymphatic system, the endocrine system, theurinary system, the reproductive system, or a combination thereof.

The polynucleotide of interest within the nucleic acid insert and/orintegrated at the target genomic locus on a chromosome can comprises agenetic modification in a coding sequence. Such genetic modificationsinclude, but are not limited to, a deletion mutation of a codingsequence or the fusion of two coding sequences.

The polynucleotide of interest within the nucleic acid insert and/orintegrated at the target genomic locus on a chromosome can comprise apolynucleotide encoding a mutant protein. In one embodiment, the mutantprotein is characterized by an altered binding characteristic, alteredlocalization, altered expression, and/or altered expression pattern. Inone embodiment, the polynucleotide of interest within the nucleic acidinsert and/or integrated at the genomic target locus on a chromosomecomprises at least one disease allele. In such instances, the diseaseallele can be a dominant allele or the disease allele is a recessiveallele. Moreover, the disease allele can comprise a single nucleotidepolymorphism (SNP) allele. The polynucleotide of interest encoding themutant protein can be from any organism, including, but not limited to,a mammal, a non-human mammal, rodent, mouse, rat, a human, a monkey, anagricultural mammal or a domestic mammal polynucleotide encoding amutant protein.

The polynucleotide of interest within the nucleic acid insert and/orintegrated at the target genomic locus on a chromosome can also comprisea regulatory sequence, including for example, a promoter sequence, anenhancer sequence, a transcriptional repressor-binding sequence, or atranscriptional terminator sequence. In specific embodiments, thepolynucleotide of interest within the nucleic acid insert and/orintegrated at the target genomic locus on a chromosome comprises apolynucleotide having a deletion of a non-protein-coding sequence, butdoes not comprise a deletion of a protein-coding sequence. In oneembodiment, the deletion of the non-protein-coding sequence comprises adeletion of a regulatory sequence. In another embodiment, the deletionof the regulatory element comprises a deletion of a promoter sequence.In one embodiment, the deletion of the regulatory element comprises adeletion of an enhancer sequence. Such a polynucleotide of interest canbe from any organism, including, but not limited to, a mammal, anon-human mammal, rodent, mouse, rat, a human, a monkey, an agriculturalmammal or a domestic mammal polynucleotide encoding a mutant protein.

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise, if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Many modificationsand other embodiments of the methods and compositions set forth hereinwill come to mind to one skilled in the art to which this methods andcompositions pertains having the benefit of the teachings presented inthe foregoing descriptions and the associated drawings. Therefore, it isto be understood that the methods and compositions are not to be limitedto the specific embodiments disclosed and that modifications and otherembodiments are included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The described invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indicating the scope of theinvention.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Generation of Human iPS Cells

This example describes the generation of human iPS cells fromnon-pluripotent human cells. A sample protocol is shown in Table 2.PiggyBac (System Biosciences) vectors (PB-600A_CAGGGS Bst XI (0.64μg/μL) and PB-200 (0.99 μg/μL) comprising the genes that encode fourreprogramming factors (hOct4, hSox2, hKLF-4, hMYC) operably linked to aCM7 promoter were introduced into neonatal human foreskin fibroblastsusing RED and BLUE GeneIn™ transfection reagents (GlobalStem). Thetransfected cells were incubated on NuFF1 feeder cells in E7 medium(Life Technologies) to allow for incorporation of the vectors andexpression of the reprogramming factors. E7 medium comprised DMEM/F-12,NaHCO₃, L-ascorbic acid, insulin, transferrin, selenium, and FGF-2.

Puromycin selection began 10 days after transfection using 2 μg/mLpuromycin in E7 medium. At day 21, colonies were selected and culturedin mTeSR™ medium (mTeSR™ 1 medium from STEMCELL Technologies), whichcomprised DMEM/F-12, NaHCO₃, L-ascorbic acid, insulin, transferrin,selenium, FGF-2, TGF-β1, glutathione, L-glutamine, defined lipids,thiamine, trace elements B and C, β-mercaptoethanol, bovine serumalbumin, pipecolic acid, lithium chloride, and GABA. A comparison of themTeSR™ medium and E7 medium components is shown in Table 3. At days 29to 57, cells were propagated and passaged in mTeSR™ medium untilreaching ˜50% confluent in 6 well plates. At days 65 to 73, propagationand passage continued using mTeSR™ medium and Gentle Cell DissociationReagent (Stem Cell Technologies). At day 76, medium was changed to lowosmolality VG2i medium for further propagation, passage, and maintenanceof the cells comprising naïve or naïve-looking hiPSCs. The timing ofpassage in the above method was determined by cell morphology.

TABLE 2 Sample protocol for generation of human iPS cells. Day ActionMedium  0 Plate NuFF1 cells in 6-well plates (1 × 10⁵ per well)Fibroblast medium  1 piggyBac transfection E7 Medium 10 Start puromycinselection (2 ug/mL) 21 Pick up colonies by cutting mTeSR 29-57 Passagecells until reach ~50% of well (passage by Medium Gentle CellDissociation Reagent) 65-73 Continue passaging cells and freezing 2/3when passage (passage by Gentle Cell Dissociation Reagent) 76 Changemedium to VG2i VG2i

TABLE 3 Comparison of components of mTeSR and E7 media. Component mTeSRE7 DMEM/F-12 + + L-Ascorbic Acid + + Insulin + + Transferrin + +Selenium + + FGF2 + + TGFβ1 + Glutathione + L-Glutamine + DefinedLipids + Thiamine + Trace Elements B + Trace Elements C +B-Mercaptoethanol + Albumin (BSA) + Pipecolic Acid + LiCl + GABA +

Example 2 LTVEC Targeting in Human iPS Cells

This example describes the use of LTVEC targeting in human iPS cells. Asshown in FIG. 1, we introduced by electroporation into human iPS cellspropagated in VG2i medium the following nucleic acid molecules: (1) anLTVEC (0.67 μg); (2) a plasmid encoding a Cas9 endonuclease (5 μg); and(3) a plasmid encoding a CRISPR single guide RNA (gRNA) (10 μg). In oneset of samples, the Cas9 and gRNA were excluded. Specifically, 3×10⁶cells were electroporated at a voltage of 700V, a capacitance of 25 uF,and a resistance of 400 ohms The LTVEC comprised a 16.7 kb nucleic acidcomprising mouse Adam6a and Adam6b genes flanked by homology armscontaining 34 kb and 105 kb of genomic DNA derived from genomic regionsthat flank the 4.1 kb sequence of the human ADAM6 locus intended fordeletion. The LTVEC also carried a drug selection cassette that directsthe expression of an enzyme that imparts resistance to an antibioticdrug (hygromycin). The human ADAM6 gRNA used had the following sequence:GTATAGCCCTGTTACACATT (SEQ ID NO: 1).

Cells that took up the LTVEC and incorporated it into their genomes wereable to grow and form colonies on a GELTREX™-coated tissue culture dishin a growth medium containing the antibiotic drug. Because we introduced500 to 1,000 times more CRISPR/Cas9-encoding nucleic molecules thanLTVEC molecules, most of the LTVEC-containing drug resistant coloniesalso contained, at least transiently, the CRISPR/Cas9 components. Wepicked drug resistant colonies and screened them by the loss-of-allelemethod (Valenzuela et al. (2003) Nat. Biotech. 21:652-660; Frendewey etal. (2010) Methods Enzymol. 476:295-307; incorporated herein byreference in their entireties) to identify clones that had the correctlytargeted allele.

The results of the CRISPR/Cas9-assisted LTVEC targeting of the ADAM6locus are shown in Table 4.

TABLE 4 CRISPR/Cas9-assisted LTVEC targeting. Targeting ConditionTargeting Efficiency LTVEC Only 3.1% LTVEC + CRISPR 7.3%

When the LTVEC alone was introduced into human iPS cells, a targetingefficiency of 3.1% was observed. In contrast, combining the LTVEC withCas9 guided by the ADAM6 gRNA resulted in a targeting efficiency of7.3%.

Example 3 Effect of Low Osmolality Medium on Human iPS Cell Morphology.

This example describes the effect of salt concentration, ionic strength,and/or osmolality on the pluripotency state of human iPS cells inculture. Human iPS cells were cultured on a MATRIGEL™ or GELTREX™substrate in a medium described in Table 5 (final hLIF concentration of100 U/mL; final CHIR99021 concentration of 3 μM, and final PD0325901concentration of 0.5 μM) or in mTeSR™-hLIF medium.

TABLE 5 Medium for iPS cell culture. Component Amount (v/v) Base Medium24.75 F-12 Medium 24.75 N2 ® Supplement 0.5 Neurobasal ® Medium 49B-27 ® Supplement 1 Penicillin/Streptomycin 1 L-Glutamine (200 mM) 12-Mercaptoethanol (55 mM) 0.1836 hLIF (1 × 10⁴ units/mL) 0.001 CHIR99021(10 mM) 0.03 PD0325901 (10 mM) 0.005

TABLE 6 Osmolality of medium and medium components. Medium or MediumComponent Osmolality (mOsm/kg) 2i Medium 261 VG2i Medium 233 Neurobasal216 DMEM/F-12 305 DMEM 340 F-12 290 VG-DMEM 200

When the base medium used was DMEM, this medium was referred to as 2imedium. When the base medium used was VG-DMEM, this low osmolalitymedium was referred to as VG2i medium. The osmolality of VG2i medium(233 mOsm/kg) is lower than the osmolality of traditional 2i medium (261mOsm/kg). Table 6 shows the osmolalities for these media as well as theosmolality of different base media and media components.

As shown in FIG. 2, human iPS cells cultured on MATRIGEL™ in 2i mediumfor a period of 8 days (FIG. 2A) or 12 days (FIG. 2B) displayed amorphology characteristic of iPS cells in a primed state, particularlygrowth in an epithelial monolayer and the appearance of apico-basalpolarity.

mTeSR™-hLIF medium and VG2i medium were further evaluated for theireffects on the morphology and pluripotency state of human iPS cells. Inthis study, human iPS cells were cultured on MATRIGEL™ or NuFF feedercells in mTeSR™-hLIF medium (FIGS. 3A and 3C) or in VG2i medium (FIGS.3B and 3D) for a period of 6 days. When cultured in mTeSR™-hLIF mediumon MATRIGEL™ or NuFF feeder cells, human iPS cells displayed amorphology characteristic of a primed pluripotency state, particularlygrowth in an epithelial monolayer and the appearance of apico-basalpolarity. Some cells cultured in mTeSR™-hLIF medium began to display amorphology characterized by three-dimensional clumping. By contrast,when cultured in VG2i medium on MATRIGEL™ or NuFF feeder cells, thehuman iPS cells displayed a morphology characteristic of a naïvepluripotency state, particularly growth in round, dome-shaped coloniesand a lack of apico-basal polarity.

Example 4 Effect of Low Osmolality Medium on the Expression ofPluripotency Markers in Human iPS Cells

This example describes the effect of salt concentration, ionic strength,and/or osmolality on the expression of pluripotency markers in human iPScells that have been reprogrammed from a primed state to a naïve state.Following 24 days of culture in VG2i medium on a MATRIGEL™ substrate,reprogrammed naïve human iPS cells were stained for the expression ofalkaline phosphatase or NANOG. It was observed that the reprogrammedcells strongly expressed both alkaline phosphatase (FIG. 4A) and NANOG(FIGS. 4B and 4C), which are indicative of a naïve pluripotency state.

Example 5 Effect of Low Osmolality Medium on Enzymatic Dissociation andSubculture of Human iPS Cells

In this example, human iPS cells that were reprogrammed to a naïve stateusing low osmolality VG2i medium were enzymatically dissociated usingtrypsin to create a single cell suspension (FIG. 5A). The cellsuspension was passaged onto new GELTREX™-coated plates for subculturein VG2i medium. It was observed after 1 day (FIG. 5B) and 4 days (FIG.5C) that the subcultured cells continued to display a morphologycharacteristic of cells in a naïve pluripotency state. Particularly, thecells grew as rounded dome-shaped colonies and did not exhibit anapico-basal polarity. It was notable that enzymatic dissociation couldbe performed in the absence of a ROCK inhibitor, which is typicallynecessary to prevent activation of pro-apoptotic pathways. This suggeststhat pro-apoptotic pathways are not as strongly activated duringenzymatic dissociation and subculture in naïve human iPS cells culturedunder the conditions identified herein. In addition, human iPS cellscultured in VG2i and passaged as single cells following enzymaticdissociation with trypsin maintain a normal karyotype. Two passage 10human iPS cells derived from different clones and produced followingdissociation with trypsin to create a single-cell suspension werekaryotyped, and both had a normal 46 XY karyotype (FIGS. 6A and 6B).

1. An in vitro culture comprising: (a) a population of hiPSCs; and (b) alow osmolality medium comprising a base medium and supplements, whereinthe low osmolality medium comprises: (i) a leukemia inhibitory factor(LIF) polypeptide; (ii) a glycogen synthase kinase 3 (GSK3) inhibitor;and (iii) a MEK inhibitor; wherein the base medium has an osmolality ofabout 180 mOsm/kg to about 250 mOsm/kg.
 2. The in vitro culture of claim1, wherein the hiPSCs: (a) comprise naïve or naïve-looking hiPSCs; (b)express one or more pluripotency markers; (c) display a morphologycharacterized by compact dome-shaped colonies; (d) can differentiateinto cells of any one of the endoderm, ectoderm, or mesoderm germlayers; (e) have a doubling time of between about 16 hours and about 24hours; or (f) any combination of (a) to (e).
 3. The in vitro culture ofclaim 1, wherein the hiPSCs have a normal karyotype.
 4. The in vitroculture of claim 2, wherein the pluripotency markers comprise NANOG,alkaline phosphatase, or a combination thereof.
 5. The in vitro cultureof claim 1, wherein the hiPSCs are derived from non-pluripotent cellstransformed to express a pluripotent state.
 6. The in vitro culture ofclaim 5, wherein the transformed cells express reprogramming genescomprising Oct4, Sox2, Klf4, Myc, or any combination thereof.
 7. The invitro culture of claim 5, wherein the transformed cells comprise primedhiPSCs.
 8. The in vitro culture of claim 5, wherein the transformedcells are first cultured in a high osmolality medium prior to culturingin the low osmolality medium, wherein the high osmolality mediumcomprises bFGF.
 9. The in vitro culture of claim 8, wherein the highosmolality medium has an osmolality of at least about 290 mOsm/kg. 10.The in vitro culture of claim 8, wherein: (a) the transformed cells arefirst cultured in the high osmolality medium until they expresscharacteristics of a naïve or naïve-looking state; (b) the transformedcells are first cultured in the high osmolality medium for a period ofabout two months; (c) the transformed cells are first cultured in thehigh osmolality medium until they display a morphology characterized bythree-dimensional cell clumps; or (d) a combination thereof.
 11. The invitro culture of claim 1, wherein the base medium comprises NaCl atabout 3 mg/ml, sodium bicarbonate at about 2.2 mg/mL, and has anosmolality of about 200 mOsm/kg.
 12. The in vitro culture of claim 1,wherein the base medium comprises glucose at about 4.5 mg/mL.
 13. The invitro culture of claim 1, wherein the low osmolality medium has anosmolality of about 200 mOsm/kg to about 250 mOsm/kg.
 14. The in vitroculture of claim 13, wherein the low osmolality medium has an osmolalityof about 233 mOsm/kg.
 15. The in vitro culture of claim 1, wherein: (a)the supplements comprise: (i) F-12 medium; (ii) N2 supplement; (iii)NEUROBASAL medium; (iv) B-27 supplement; (v) L-glutamine; (vi)2-mercaptoethanol; or (vii) any combination of (i) to (vi); (b) the LIFpolypeptide is a human LIF (hLIF) polypeptide; (c) the GSK3 inhibitorcomprises CHIR99021; (d) the MEK inhibitor comprises PD0325901; or (e)any combination of (a) to (d).
 16. The in vitro culture of claim 1,wherein the low osmolality medium comprises inhibitors consistingessentially of a glycogen synthase kinase 3 (GSK3) inhibitor and a MEKinhibitor.
 17. The in vitro culture of claim 1, wherein the lowosmolality medium comprises base medium at about 24.75% (v/v), F-12medium at about 24.75% (v/v), N2 supplement at about 0.5% (v/v),NEUROBASAL medium at about 49% (v/v), B-27 supplement at about 1% (v/v),L-glutamine at about 2 mM, 2-mercaptoethanol at about 0.1 mM, hLIF atabout 100 units/mL, CHIR99021 at about 3 μM, and PD0325901 at about 0.5μM.
 18. The in vitro culture of claim 1, wherein the low osmolalitymedium does not comprise one or more of the following: bFGF supplement;TGF-β1 supplement; JNK inhibitor; p38 inhibitor; ROCK inhibitor; and PKCinhibitor.
 19. The in vitro culture of claim 18, wherein the lowosmolality medium does not comprise bFGF supplement.
 20. The in vitroculture of claim 1, wherein the hiPSCs are cultured on MATRIGEL, NuFFfeeder cells, or GELTREX.
 21. The in vitro culture of claim 1, whereinthe hiPSCs can be enzymatically dissociated into a single-cellsuspension and subcultured.
 22. The in vitro culture of claim 21,wherein the enzymatic dissociation: (a) is performed using trypsin; (b)is performed in the absence of a ROCK inhibitor; or (c) a combinationthereof.
 23. The in vitro culture of claim 22, wherein the subculturedhiPSCs: (a) continue to express the one or more pluripotency markers;(b) maintain a naïve or naïve-looking state and display a morphologycharacterized by compact dome-shaped colonies; or (c) a combinationthereof.
 24. The in vitro culture of claim 22, wherein the subculturedhiPSCs maintain a normal karyotype.
 25. A population of hiPSCs made ormaintained in a low osmolality medium comprising a base medium andsupplements, wherein the low osmolality medium comprises: (a) a leukemiainhibitory factor (LIF) polypeptide; (b) a glycogen synthase kinase 3(GSK3) inhibitor; and (c) a MEK inhibitor; wherein the base medium hasan osmolality of about 180 mOsm/kg to about 250 mOsm/kg.
 26. Thepopulation of claim 25, wherein the hiPSCs: (a) comprise naïve ornaïve-looking hiPSCs; (b) express one or more pluripotency markers; (c)display a morphology characterized by compact dome-shaped colonies; (d)can differentiate into cells of any one of the endoderm, ectoderm, ormesoderm germ layers; (e) have a doubling time of between about 16 hoursand about 24 hours; or (f) any combination of (a) to (e).
 27. Thepopulation of claim 25, wherein the hiPSCs have a normal karyotype. 28.The population of claim 26, wherein the pluripotency markers compriseNANOG, alkaline phosphatase, or a combination thereof.
 29. Thepopulation of claim 25, wherein the hiPSCs are derived fromnon-pluripotent cells transformed to express a pluripotent state. 30.The population of claim 29, wherein the transformed cells expressreprogramming genes comprising Oct4, Sox2, Klf4, Myc, or any combinationthereof.
 31. The population of claim 29, wherein the transformed cellscomprise primed hiPSCs.
 32. The population of claim 29, wherein thetransformed cells are first cultured in a high osmolality medium priorto culturing in the low osmolality medium, wherein the high osmolalitymedium comprises bFGF.
 33. The population of claim 32, wherein the highosmolality medium has an osmolality of at least about 290 mOsm/kg. 34.The population of claim 32, wherein: (a) the transformed cells are firstcultured in the high osmolality medium until they expresscharacteristics of a naïve or naïve-looking state; (b) the transformedcells are first cultured in the high osmolality medium for a period ofabout two months; (c) the transformed cells are first cultured in thehigh osmolality medium until they display a morphology characterized bythree-dimensional cell clumps; or (d) a combination thereof.
 35. Thepopulation of claim 25, wherein the base medium comprises NaCl at about3 mg/ml, sodium bicarbonate at about 2.2 mg/mL, and has an osmolality ofabout 200 mOsm/kg.
 36. The population of claim 25, wherein the basemedium comprises glucose at about 4.5 mg/mL.
 37. The population of claim25, wherein the low osmolality medium has an osmolality of about 200mOsm/kg to about 250 mOsm/kg.
 38. The population of claim 37, whereinthe low osmolality medium has an osmolality of about 233 mOsm/kg. 39.The population of claim 25, wherein: (a) the supplements comprise: (i)F-12 medium; (ii) N2 supplement; (iii) NEUROBASAL medium; (iv) B-27supplement; (v) L-glutamine; (vi) 2-mercaptoethanol; or (vii) anycombination of (i) to (vi); (b) the LIF polypeptide is a human LIF(hLIF) polypeptide; (c) the GSK3 inhibitor comprises CHIR99021; (d) theMEK inhibitor comprises PD0325901; or (e) any combination of (a) to (d).40. The population of claim 25, wherein the low osmolality mediumcomprises inhibitors consisting essentially of a glycogen synthasekinase 3 (GSK3) inhibitor and a MEK inhibitor.
 41. The population ofclaim 25, wherein the low osmolality medium comprises base medium atabout 24.75% (v/v), F-12 medium at about 24.75% (v/v), N2 supplement atabout 0.5% (v/v), NEUROBASAL medium at about 49% (v/v), B-27 supplementat about 1% (v/v), L-glutamine at about 2 mM, 2-mercaptoethanol at about0.1 mM, hLIF at about 100 units/mL, CHIR99021 at about 3 μM, andPD0325901 at about 0.5 μM.
 42. The population of claim 25, wherein thelow osmolality medium does not comprise one or more of the following:bFGF supplement; TGF-β1 supplement; JNK inhibitor; p38 inhibitor; ROCKinhibitor; and PKC inhibitor.
 43. The population of claim 42, whereinthe low osmolality medium does not comprise bFGF supplement.
 44. Thepopulation of claim 25, wherein the hiPSCs are cultured on MATRIGEL,NuFF feeder cells, or GELTREX.
 45. The population of claim 25, whereinthe hiPSCs can be enzymatically dissociated into a single-cellsuspension and subcultured.
 46. The population of claim 45, wherein theenzymatic dissociation: (a) is performed using trypsin; (b) is performedin the absence of a ROCK inhibitor; or (c) a combination thereof. 47.The population of claim 46, wherein the subcultured hiPSCs: (a) continueto express the one or more pluripotency markers; (b) maintain a naïve ornaïve-looking state and display a morphology characterized by compactdome-shaped colonies; or (c) a combination thereof.
 48. The populationof claim 46, wherein the subcultured hiPSCs maintain a normal karyotype.49. A method for making a population of human induced pluripotent stemcells (hiPSCs), the method comprising culturing in vitro a population ofnon-pluripotent cells, transformed to express a pluripotent state, in alow osmolality medium comprising a base medium and supplements, whereinthe low osmolality medium comprises: (a) a leukemia inhibitory factor(LIF) polypeptide; (b) a glycogen synthase kinase 3 (GSK3) inhibitor;and (c) a MEK inhibitor; wherein the base medium has an osmolality ofabout 180 mOsm/kg to about 250 mOsm/kg. 50.-72. (canceled)
 73. A methodfor maintaining a population of hiPSCs in an in vitro culture, themethod comprising culturing the population of hiPSCs in a low osmolalitymedium comprising a base medium and supplements, wherein the lowosmolality medium comprises: (a) a leukemia inhibitory factor (LIF)polypeptide; (b) a glycogen synthase kinase 3 (GSK3) inhibitor; and (c)a MEK inhibitor; wherein the base medium has an osmolality of about 180mOsm/kg to about 250 mOsm/kg. 74.-96. (canceled)
 97. A method formodifying a target genomic locus in a hiPSC, comprising: (a) introducinginto the hiPSC a targeting vector comprising an insert nucleic acidflanked by 5′ and 3′ homology arms corresponding to 5′ and 3′ targetsites at the target genomic locus; and (b) identifying a geneticallymodified hiPSC comprising in its genome the insert nucleic acidintegrated at the target genomic locus; wherein the hiPSC is cultured ina low osmolality medium comprising a base medium and supplements,wherein the low osmolality medium comprises: (i) a leukemia inhibitoryfactor (LIF) polypeptide; (ii) a glycogen synthase kinase 3 (GSK3)inhibitor; and (iii) a MEK inhibitor; wherein the base medium has anosmolality of about 180 mOsm/kg to about 250 mOsm/kg.
 98. -127.(canceled)
 128. A method for modifying a target genomic locus in ahiPSC, comprising: (a) introducing into the hiPSC one or more nucleaseagents that induces one or more nicks or double-strand breaks at arecognition site at the target genomic locus; and (b) identifying atleast one cell comprising in its genome a modification at the targetgenomic locus; wherein the hiPSC is cultured in a low osmolality mediumcomprising a base medium and supplements, wherein the low osmolalitymedium comprises: (i) a leukemia inhibitory factor (LIF) polypeptide;(ii) a glycogen synthase kinase 3 (GSK3) inhibitor; and (iii) a MEKinhibitor; wherein the base medium has an osmolality of about 180mOsm/kg to about 250 mOsm/kg. 129.-154. (canceled)